Sphingoid polyalkylamine conjugates for vaccination

ABSTRACT

The present invention concerns the use of a sphingoid-polyalkylamine conjugate as a capturing agent of biologically active molecules, such as antigens. In a particular embodiment, the spinogid-polyalkylamines are used for the preparation of pharmaceutical composition for modulating the immune response of a subject. Other aspects of the invention concern methods for modulating the immune response of a subject by the use of the conjugate, complexes comprising, the sphingoid-polyalkylamine conjugate in combination with a biologically active molecule capable of modulating an immune response of a subject, compositions comprising the conjugate as well as kits making use of said conjugate. A preferred conjugate according to the invention is N palmitoyl D-erythro sphingosyl 1 carbamoyl spermine.

FIELD OF THE INVENTION

The present invention concerns vaccination making use of sphingolipids'polyalkylamine conjugates for effective delivery of biologically activematerials, in particular, antigenic molecules.

LIST OF PRIOR ART

The following is a list of prior art which is considered to be pertinentfor describing the state of the art in the field of the invention.

-   U.S. Pat. No. 5,334,761: “Cationic lipids”;-   US 2001/048939: “Cationic reagents of transfection”;-   U.S. Pat. No. 5,659,011: “Agents having high nitrogen content and    high cationic charge based on dicyanimide dicyandiamide or guanidine    and inorganic ammonium salts”;-   U.S. Pat. No. 5,674,908: “Highly packed polycationic ammonium,    sulfonium and phosphonium lipids”;-   U.S. Pat. No. 6,281,371: “Lipopolyamines, and the preparation and    use thereof”;-   U.S. Pat. No. 6,075,012: “Reagents for intracellular delivery of    macromolecules”;-   U.S. Pat. No. 5,783,565: “Cationic amphiphiles containing spermine    or spermidine cationic group for intracellular delivery of    therapeutic molecules”;-   Marc Antoniu Ilies & Alexandru T. Balaban, Expert Opin. Ther.    Patents. 11(11):1729-1752 (2001);-   Miller A D. Chem. Int. Ed. Eng. 37:1768-1785 (1998);-   Nakanichi T. et al. J. Control Release 61:233-240 (1999);-   Brunel F. et al. Vaccine 17:2192-2193 (1999);-   Guy B. et al. Vaccine 19:1794-1805 (2001);-   Lima K M et al. Vaccine 19:3518-3525 (2001).

BACKGROUND OF THE INVENTION

Many natural biological molecules and their analogues, includingproteins and polynucleotides, foreign substances and drugs, which arecapable of influencing cell function at the sub-cellular or molecularlevel are preferably incorporated within the cell in order to producetheir effect. For these agents the cell membrane presents a selectivebarrier which is impermeable to them. The complex composition of thecell membrane comprises phospholipids, glycolipids, and cholesterol, aswell as intrinsic and exrinsic proteins, and its functions areinfluenced by cytoplasmic components which include Ca⁺⁺ and other metalions, anions, ATP, microfilaments, microtubules, enzymes, andCa⁺⁺-binding proteins, also by the extracellular glycocalyx(proteoglycans, glycose aminoglycans and glycoproteins). Interactionsamong structural and cytoplasmic cell components and their response toexternal signals make up transport processes responsible for themembrane selectivity exhibited within and among cell types.

Successful delivery of agents not naturally taken up by cells into cellshas also been investigated. The membrane barrier can be overcome byassociating agents in complexes with lipid formulations closelyresembling the lipid composition of natural cell membranes. Theseformulations may fuse with the cell membranes on contact, or what ismore common, taken up by pynocytosis, endocytosis and/or phagocytosis.In all these processes, the associated substances are delivered in tothe cells.

Lipid complexes can facilitate intracellular transfers also byovercoming charge repulsions between the cell surface, which in mostcases is negatively charged. The lipids of the formulations comprise anamphipathic lipid, such as the phospholipids of cell membranes, and formvarious layers or aggregates such as micelles or hollow lipid vesicles(liposomes), in aqueous systems. The liposomes can be used to entrap thesubstance to be delivered within the liposomes; in other applications,the drug molecule of interest can be incorporated into the lipid vesicleas an intrinsic membrane component, rather than entrapped into thehollow aqueous interior, or electrostatically attached to aggregatesurface. However, most phospholipids used are either zwiterionic(neutral) or negatively charged.

An advance in the area of intracellular delivery was the discovery thata positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),in the form of liposomes, or small vesicles, could interactspontaneously with DNA to form lipid-DNA complexes which are capable ofadsorbing to cell membranes and being taken up by the cells either byfusion or more probably by adsorptive endocytosis, resulting inexpression of the transgene [Felgner, P. L. et al. Proc. Natl. Acad.Sci., USA 84:7413-7417 (1987) and U.S. Pat. No. 4,897,355 to Eppstein,D. et al.]. Others have successfully used a DOTMA analogue,1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP) in combinationwith a phospholipid to form DNA-complexing vesicles. The Lipofectin™reagent (Bethesda Research Laboratories, Gaithersburg, Md.), aneffective agent for the delivery of highly anionic polynucleotides intoliving tissue culture cells, comprises positively charged liposomescomposed of positively charged lipid DOTMA and a neutral lipid dioleylphosphatidyl ethanol amine (DOPE) referred to as helper lipids. Theseliposomes interact spontaneously with negatively charged nucleic acidsto form complexes, referred to as lipoplexes. When excess of positivelycharged liposomes over DNA negative charges are used, the net charge onthe resulting complexes is also positive. Positively charged complexesprepared in this way spontaneously attach to negatively charged cellsurfaces or introduced into the cells either by adsorptive endocytosisor fuse with the plasma membrane, both processes deliver functionalpolynucleotide into, for example, tissue culture cells. DOTMA and DOTAPare good examples for monocationic lipids. [Illis et al. 2001, ibid.]

Multivalent cations by themselves (including polyamines, inorganic saltsand complexes and dehydrating solvents) have also been shown tofacilitate delivery of macromolecules into cells. In particular,multivalent cations provoke the collapse of oligo and polyanions(nucleic acids molecules, amino acid molecules and the like) to compactstructural forms, and facilitate the packaging of these polyanions intoviruses, their incorporation into liposomes, transfer into cells etc.[Thomas T. J. et al. Biochemistry 38:3821-3830 (1999)]. The smallestnatural polycations able to compact DNA are the polyamines spermidineand spermine. By attaching a hydrophobic anchor to these molecules via alinker, a new class of transfection vectors, the polycationiclipopolymers, has been developed.

Cationic lipids and cationic polymers interact electrostatically withthe anionic groups of DNA (or of any other polyanionic macromolecule)forming DNA-lipid complexes (lipoplexes) or DNA-polycation complexes(polyplexes). The formation of the complex is associated with therelease of counterions of the lipids or polymer, which is thethermodynamic driving force for lipoplex and polyplex spontaneousformation. The cationic lipids can be divided into four classes: (i)quaternary ammonium salt lipids (e.g. DOTMA (Lipofectin™) and DOTAP) andphosphonium/arsonium congeners; (ii) lipopolyamines; (iii) cationiclipids bearing both quaternary ammonium and polyamine moieties and (iv)amidinium, guanidinium and heterocyclic salt lipids.

SUMMARY OF THE INVENTION

According to one of its aspects, the present invention concerns the useof a sphingoid-polyalkylamine conjugate for the preparation of apharmaceutical composition for modulating the immune response of asubject.

According to a preferred embodiment, the sphingoid-polyalkylamineconjugate comprises a sphingoid backbone carrying, via a carbamoyl bondat least one polyalkylamine chain polyalkylamine chain.

The term sphingoid-polyalkylamine conjugate as used herein denoteschemical conjugation (linkage) between a sphingoid base (herein alsoreferred to by the term “sphingoid backbone”) and at least onepolyalkylamine chain. The conjugation between the sphingoid base and theat least one polyalkylamine chain is via a carbamoyl bond, as furtherdetailed hereinafter.

The sphingoid base/backbone, as used herein, includes, long chainaliphatic amines, containing two or three hydroxyl groups, the aliphaticchain may be saturated or unsaturated. One example of an unsaturatedsphingoid base is that containing a distinctive trans-double bond inposition 4.

The term modulating as used herein denotes any measurable regulatory orbiochemical effect exhibited by the biologically active materialdelivered by the conjugate, on a subject's immune response, includingcellular response and/or humoral response. Modulation includesinhibition or, on the other hand, stimulation or enhancement of eitheror both types of responses when the sphingoid-polyalkylamine conjugateis administered to said subject in combination with a biologicallyactive substance. The modulation preferably refers to stimulation orenhancement by a factor of two or more, relative to that elicited by thebiological active molecule administered without the conjugate. Theinvention also concerns the modulation of an immune response in caseswhen the biologically active material administered without the conjugateis substantially ineffective in producing such a response.

Yet further, modulation concerns inhibition or suppression of the immuneresponse of a subject, e.g. for the treatment of auto-immune diseases aswell as for the treatment of allergy.

Thus, the term biologically active molecule as used herein denotes anysubstance which, when administered in combination with thesphingoid-polyalkylamine conjugate has an effect on the immune system ofa subject. The biologically active material is preferably an antigenicprotein, antigenic peptide, antigenic polypeptide or antigeniccarbohydrate.

According to another aspect, the present invention concerns a method formodulating the immune response of a subject, the method comprisesproviding said subject with a sphingoid-polyalkylamine conjugatetogether with a biologically active molecule, saidsphingoid-polyalkylamine conjugate comprises a sphingoid backbonecarrying, via a carbamoyl bond at least one polyalkylamine chain.

According to yet another aspect, the present invention concerns apharmaceutical composition for modulating the immune response of asubject, the composition comprises: (i) at least onesphingoid-polyalkylamine conjugate; and (ii) at least one biologicallyactive molecule associated with said conjugate.

According to yet another embodiment, the invention provides a complexcomprising: (i) a sphingoid-polyalkylamine conjugate and (ii) abiologically active material capable of modulating an immune response ofa subject.

Finally, the invention concerns the use of

Finally, the invention concerns the use of a sphingoid-polyalkylamineconjugate as defined, as a capturing agent of biologically-activemolecules (e.g. antigenic molecules). In this context, thesphingoid-polyalkylamine conjugate may form part of a kit for capturingbiologically active molecules, preferably antigenic molecules and/orimmunostimulants, and/or immunosuppressants, the kit comprising, inaddition to said conjugate, instructions for use of same for capturingthe biologically active molecules. The conjugate in the kit may be in adry form, in which case, the kit may also include a suitable fluid withwhich the conjugate is mixed prior to use to form a suspension oremulsion or solution, or it may already be in a fluid (suspension,emulsion, solution, etc.) form. The kit may have numerous applications.For example, the kit may be used for investigating the function ofdifferent immunomodulating molecules in modulation of immune responses,for isolation of active biological molecules, and identificationthereof. Those versed in the art will know how to make use of such acapturing agent also for research purposes.

The term capturing agent as used herein refers to the conjugate beingcapable of associating with biologically active molecules, the latterhaving a negative charge, a negative dipole or a local negative charge(an area within the molecule carrying a net negative charge), by virtueof the conjugate's polycationic structure. The capturing per se involveselectrostatic interaction between the molecule to be captured, carryingsaid negative charge, negative dipole or local negative charge and thepositively charged conjugate of the invention.

The conjugate of the invention may also be used as a delivery vehicle,carrying, by capturing thereto, biologically active molecules to atarget site and into a target cell.

BRIEF DESCRIPTION OF THE FIGURES

In order to understand the invention and to see how it may be carriedout in practice, some embodiments will now be described, by way ofnon-limiting examples only, with reference to the accompanying figures,in which:

FIGS. 1A-1D show several possible chemical structures, “linear”,branched” or “cyclic” lipid like cationic (LLC) compounds which areencompass under the general definition of sphingoid-polyalkylamineconjugate of formula (I), wherein FIG. 1A shows a sphingoid backbone(ceramide) linked to a single polyalkylamine chain, FIG. 1B and FIG. 1Cshow the same sphingoid backbone linked to two polyalkylamine chains,FIG. 1D shows again the same backbone, however, in which a singlepolyalkylamine chain is linked via the two hydroxyl moieties to form acyclic polyalkylamine conjugate.

FIGS. 2A-2F show the bio-distribution and pharmacokinetics of variousfluorescently-labeled lipid formulations in the GI-▪-, lungs -♦- orspleen --- with unrecovered -♦-: FIG. 2A shows distribution of emptyDMPC:DMPG (mole ratio 9:1); FIG. 2B shows distribution of DMPC:DMNG:HN;FIG. 2C shows distribution of empty DOTAP:cholesterol; FIG. 2D showsdistribution of DOTAP:cholesterol:HN; FIG. 2E shows distribution ofempty CCS:cholesterol; and finally, FIG. 2F shows distribution ofCCS-cholesterol:HN.

FIGS. 3A-3D show bio-distribution of various ¹²⁵I-HN loaded lipidassembly formulations in the GI-▪-, lungs -♦- or spleen -⋄- withunrecovered -x-, and in particular, FIG. 3A shows bio-distribution offree ¹²⁵I-HN; FIG. 3B shows ¹²⁵I-HN loaded lipid assembly composed ofDOTAP:Cholesterol; FIG. 3C shows ¹²⁵I-HN loaded lipid assembly composedof DMPC:DMPG and FIG. 3D shows ¹²⁵I-HN loaded lipid assembly composed ofCCS:Cholesterol.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns the use of sphingoid-polyalkylamineconjugates as capturing agents for carrying biologically activemolecules which are effective in modulating the immune response of asubject.

The sphingoid-polyalkylamine conjugates are lipid-like cationic (LLC)compounds, which may be synthesized in the following manner.N-substituted long-chain bases in particular, N-substituted sphingoidsor sphingoid bases are coupled together with different polyalkylaminesor their derivatives, to form a polyalkylamine-sphingoid entity, whichis used as is, or further alkylated.

Protonation at a suitable pH or alkylation of the formedpolyalkylamine-sphingoid entity attributes to the lipid-like compounds adesired positive charge for interaction with biologically activebiological molecules to be delivered into target cells and with thetargeted cells. The sphingoid-polyalkylamine conjugates may beefficiently associated with the biologically active molecules by virtueof electrostatic interactions between the anionic character of thebiologically active molecules and the polyalkylamine moieties of theconjugate to form complexes (lipoplexes).

Alternatively, the sphingoid-polyalkylamine conjugates may formassemblies loaded with the biologically active molecules.

The sphingoid-polyalkylamine conjugate may be in the form individuallipid like molecules or in the form of an assembly. One example of asuitable assembly includes the formation of micelles or vesicles, and inparticular, liposomes. Other examples of assemblies include theformation of micelles, inverted phases, cubic phases and the like.Evidently, the sphingoid polyalkylamine conjugate may be in combinedvesicle/micelle form or any other combination of assemblies.

Lipid assembly as used herein denotes an organized collection of lipidmolecules forming inter alia, micelles and liposomes. The lipidassemblies are preferably stable lipid assemblies. Stable lipid assemblyas used herein denotes an assembly being chemically and physicallystable under storage conditions (4° C., in physiological medium) for atleast one month.

When the assemblies are in the form of vesicles (e.g. liposomes) thebiologically active molecule may be encapsulated within the vesicle,part of its lipid bilayer, or adsorbed to the surface of the vesicle (orany combination of these three options). When the assemblies aremicelles, the biologically active molecules may be inserted into theamphiphiles forming the micelles and/or associated with itelectrostatically, in a stable way.

Thus, as used herein, the terms “encapsulated in”, “contained in”,“loaded onto” or “associated with” indicate a physical attachmentbetween the conjugate and the biologically active molecule. The physicalattachment may be either containment or entrapment of the moleculewithin assemblies (e.g. (vesicles, micelles or other assemblies) formedfrom the conjugate; non-covalent linkage of the biological molecule tothe surface of such assemblies, or embedment of the biological moleculein between the sphingoid-polyalkylamine conjugates forming suchassemblies. It should be noted that due to the positive charge orpositive dipole of the sphingoid-polyalkylamine conjugate underphysiological conditions, the preferred association between theconjugate and the biologically active material is by electrostatic,dipole or acid-base interactions.

Notwithstanding the above, the invention should not be limited by theparticular type of association formed between thesphingoid-polyalkylamine conjugate and the biologically active molecule.Thus, association means any interaction between the conjugate or theassembly formed therefrom and the biologically active material which iscapable of achieving a desired therapeutic effect.

The biologically active molecule and the conjugate may be associated byany method known in the art. This includes, without being limitedthereto, post- or co-lyophilzation of the conjugate with thebiologically active molecule, or by mere mixing of preformedsphingoid-polyalkylamine conjugate with the biological molecule. Methodfor co-lyophilization are described, inter alia, in U.S. Pat. Nos.6,156,337 and 6,066,331, while methods for post-encapsulation aredescribed, inter alia, in WO03/000227, all incorporated herein byreference.

Thus, according to a first of its aspects, the present inventionconcerns the use of a sphingoid-polyalkylamine conjugate for thepreparation of a pharmaceutical composition for modulating the immuneresponse of a subject, wherein said sphingoid-polyalkylamine conjugatecomprises a sphingoid backbone carrying, via a carbamoyl bond, at leastone, and preferably one or two, polyalkylamine chains.

As indicated above, the sphingoid-polyalkylamine conjugate includes alinkage between a sphingoid backbone and at least one polyalkylaminechain, the linkage is via corresponding carbamoyl bonds. Morepreferably, the sphingoid-polyalkylamine conjugate has the generalformula (I):

wherein

R₁ represents a hydrogen, a branched or linear alkyl, aryl, alkylamine,or a group —C(O)R₅;

R₂ and R₅ represent, independently, a branched or linear C₁₀-C₂₄ alkyl,alkenyl or polyenyl groups;

R₃ and R₄ are independently a group —C(O)—NR₆R₇, R₆ and R₇ being thesame or different for R₃ and R₄ and represent, independently, ahydrogen, or a saturated or unsaturated branched or linearpolyalkylamine, wherein one or more amine units in said polyalkylaminemay be a quaternary ammonium; or

R₃ is a hydrogen; or

R₃ and R₄ form together with the oxygen atoms to which they are bound aheterocyclic ring comprising —C(O)—NR₉—[R₈—NR₉]_(m)—C(O)—, R₈ representsa saturated or unsaturated C₁-C₄ alkyl and R₉ represents a hydrogen or apolyalkylamine of the formula —[R₈—NR₉]_(n)—, wherein said R₉ or eachalkylamine unit R₈NR₉ may be the same or different in saidpolyalkylamine; and

n and m are independently an integer from 1 to 10, preferably 3 to 6;

W represents a group selected from —CH═CH—, —CH₂—CH(OH)— or —CH₂—CH₂—.

Non-limiting examples of the sphingoids or sphingoid bases which may beused according to a more specific embodiment of the invention, include,sphingosines, dihydrosphingosines, phytosphingosines,dehydrophytosphinosine and derivatives thereof. Non-limiting examples ofsuch derivatives are acyl derivatives, such as ceramide(N-acylsphingosine), dihydroceramides, phytoceramides anddihydrophytoceramides, respectively, as well as ceramines(N-alkylsphinogsine) and the corresponding derivatives (e.g.dihydroceramine, phytoceramine etc.). The suitably N-substitutedsphingoids or sphingoid bases posses free hydroxyl groups which areactivated and subsequently reacted with the polyalkylamines to form thepolyalkylamine-sphingoid entity. Non-limiting examples of activationagents are N,N′-disuccinimidylcarbonate, di- or tri-phosgene orimidazole derivatives. The reaction of these activation agents with thesphingoids or the sphingoid bases yields a succinimidyloxycarbonyl,chloroformate or imidazole carbamate, respectively, at one or bothhydroxyls. The reaction of the activated sphingoids with polyalkylaminesmay yield branched, straight (unbranched) or cyclic conjugates as shownin FIG. 1.

According to one preferred embodiment the sphingoid backbone is aceramide linked to one (FIG. 1A) or two (FIG. 1B or 1C) polyalkylaminechains, or linked via the two hydroxyl moieties to form a cyclicpolyalkylamine moiety (FIG. 1D).

The formed sphingoid-polyalkylamine conjugates may be further reactedwith methylation agents in order to form quaternary amines. Theresulting compounds are positively charged to a different degreedepending on the ratio between the quaternary, primary and/or secondaryamines within the formed conjugates. As such, thesphingoid-polyalkylamine conjugate exists as quaternized nitrogen saltincluding, but not limited to, quaternary ammonium chloride, aquaternary ammonium iodide, a quaternary ammonium fluoride, a quaternaryammonium bromide, a quaternary ammonium oxyanion and a combinationthereof.

The sphingoid-polyalkylamine conjugate is preferably used in combinationwith a biologically active molecule. The biologically active material isany molecule which when administered with the sphingoid-polyalkylamineconjugate has an effect on the immune system of a subject, according toone embodiment, a stimulating or enhancing effect. The effect ispreferably by a factor of two or more relative to the effect, if any, ofthe biologically active molecule, when provided to a subject withoutsaid conjugate.

According to one embodiment, the biologically active material is aprotein, polypeptide, peptide, or carbohydrate. Specifically, thebiologically active molecule may be an immunomodulator, includingantigenic protein or antigenic peptide, immunostimulants and/orimmunosuppressants. Antigenic proteins and peptides, immunostimulantsand immunosuppressants are all well known in the art. Preferably, thebiologically active protein or peptide or carbohydrate has at aphysiological pH either a net negative dipole moment, a net negativecharge or contains at least one region having a net negative chargenegatively charged.

According to yet another embodiment, the biologically active material isa nucleic acid molecule, such as oligodeoxynucleotides (ODN).

A preferred weight ratio between the sphingoid-polyalkylamine conjugateand biologically active material is 1000:1 to 1:1 weight ratio.

The sphingoid-polyalkylamine conjugate may also be combined with otheractive substances known to be used in combination with antigenicmolecules. Such substances include, for example, immunostimulatingagents (also known by the term “immunostimulant” or “adjuvant”). Thisincludes any substance that when added to a vaccine it improves theimmune response so that less vaccine is needed to produce a greaterresponse. The immunostimulating agent may be provided together with theconjugate/biologically active material, or within a specified timeinterval (e.g. several hours or days before or after the administrationof the conjugate/biologically active molecule).

Preferred immunostimulating agents include, without being limitedthereto, cytokines, such as interleukins (L-2, IL-10, IL-12, IL-15,IL-18), interferons (IFN alpha, beta, gamma), oligodeoxynucleotides(ODN), toxins (e.g. cholera toxin (CT), staphylococcal enterotoxin B(SEB)) heat label E. Coli enerotoxin (HLT) as well as any otheradjuvants known to be used in the art for enhancing or stimulating theimmune response to an antigenic molecule.

The assemblies may include the sphingoid-polyalkylamine conjugate(non-methylated or methylated) as the sole lipid-like ingredient, or becombined with other helper lipid substances. Such helper lipidsubstances may include non-cationic lipids like DOPE, DOPC, DMPC,Cholesterol, oleic acid or others at different mole ratios to thelipid-like compound. Cholesterol is one preferred added substance for invivo application while DOPE may be a preferred helper lipid for in vitroapplications. In this particular embodiment the mole ratio ofcholesterol to cationic lipid is within the range of 0.01-1.0 andpreferably 0.1-0.4.

The assemblies may also include enhancers (as known in the art, such asCaCl₂ and soluble polyalkylamines.

Other components which may be included in the lipid assembly, and whichare known to be used in structures of the like, are steric stabilizers.One example of a commonly used steric stabilizer is the family oflipopolymers, e.g. polyethylene glycol derivatized lipids (PEG-lipidconjugate). This family of compounds are know, inter alia, to increase(extend) the circulation time of lipids.

According to one embodiment, the formed liposomes may be shaped asunsized heterogeneous and heterolamellar vesicles (UHV) having adiameter of about 50-5000 nm. The formed UHV, may be downsized andconverted to large (more homogenous) unilamellar vesicles (LUV) having adiameter of about 50-100 nm by further processing. The structure anddimensions of the vesicles, e.g. their shape and size may have importantimplications on their efficiency as vehicles for delivery of the activebiological entities to the target, i.e. these determine their deliveryproperties.

A preferred group of polyalkylamine chains forming part of thesphingoid-polyalkylamine conjugate have been structurally definedhereinabove in connection with formula (I). According to thisembodiment, the polyalkylamine chains, which may be the same ordifferent in the conjugate of formula (I), are selected from spermine,spermidine, a polyalkylamine analog or a combination of same thereof.The term polyalkylamine analog is used to denote any polyalkylaminechain, and according to one embodiment denotes a polyalkylaminecomprising 1 to 10 amine groups, preferably from 3- to 6 and morepreferably 3 or 4 amine groups. Each alkylamine within thepolyalkylamine chain may be the same or different and may be a primary,secondary, tertiary or quaternary amine.

The alkyl moiety, which may be the same or different within thepolyalkylamine chain, is preferably a C₁-C₆ aliphatic repeating unit.Some non-limiting examples of polyalkylamine s include spermidine,N-(2-aminoethyl)-1,3-propane-diamine, 3,3′-iminobispropylamine, spermineand bis(ethyl) derivatives of spermine, polyethyleneimine.

The most preferred sphingoid-polyalkylamine conjugate according to theinvention is N-palmitoyl D-erythro sphingosyl carbamoyl-spermine (CCS).This conjugate includes a ceramide linked via a carbamoyl bond tospermine.

The sphingoid-polyalkylamine conjugate according to the invention ispreferably used for the preparation of a vaccine.

According to one embodiment, the sphingoid-polyalkylamine conjugate, andpreferably the CCS, is used for the preparation of an influenza vaccine.In this particular embodiment, the biologically active material isderived from the influenza virus or a biologically active analog of amolecule derived from influenza virus. Such analogs include anysubstance which includes an influenza derived antigenic fragment whichelicits an immune response.

A specific influenza derived antigenic material is the hemagglutinin(HA) and neuraminidase (NA) molecules, the combination is referred to asHN.

The present invention also concerns a method for modulating the immuneresponse of a subject, the method comprises treating said subject withthe sphingoid-polyalkylamine conjugate together with a biologicallyactive material.

The combined treatment includes administration of thesphingoid-polyalkylamine conjugate and biologically active materialeither together, or within a predefined time interval, such as severalhours or several days (optionally in combination with animmunostimulant). However, according to a preferred embodiment, theconjugate and biologically active material are mixed together prior toadministration to the subject.

Administration of the sphingoid-polyalkylamine conjugate together withthe biologically active material concerns another aspect of theinvention. Accordingly, there is provided a pharmaceutical compositioncomprising a physiologically acceptable carrier and an effective amountof the sphingoid-polyalkylamine conjugate together with the biologicallyactive material. The pharmaceutical composition optionally comprises animmunostimulant.

The sphingoid-polyalkylamine conjugated in combination with thebiologically active material may be administered and dosed in accordancewith good medical practice, taking into account the clinical conditionof the individual patient, the site and method of administration,scheduling of administration, patient age, sex, body weight and otherfactors known to medical practitioners.

The “effective amount” for purposes herein denotes an amount which iseffective to modulate (enhance or stimulate, as defined above) thesubject's immune response relative to the effect obtained when thebiologically active material is provide to the subject without thesphingoid-polyalkylamine conjugate. Preferably, the amount is effectiveto achieve effective immunization of a subject against a specificdisease or disorder.

Notwithstanding the above, the amount may be effective to achievesuppression or inhibition of the immune response, e.g. for the purposeof treating allergy or autoimmune responses.

The composition of the invention comprising the sphingoid-polyalkylamineconjugate associated with the biologically active material may beadministered in various ways: Non-limiting examples of administrationroutes include oral, subcutaneous (s.c.), parenteral includingintravenous (i.v.), intra-arterial (i.a.), intramuscular (i.m.),intraperitoneal (i.p.)′ intrarectal (i.r.) and intranasal (i.n.)administration, as well as by infusion techniques to the eyeintraocular. Preferably modes of administration are the intranasal orintramuscular administrations.

The physiologically acceptable carrier according to the inventiongenerally refers to inert, non-toxic solid or liquid substancespreferably not reacting with the biologically active material or withthe conjugate and which is required for the effective delivery of theconjugate with the biologically active molecule.

Non-limiting examples of physiologically acceptable carrier includewater, saline, 5% dextrose (glucose), 10% sucrose etc., either alone orwith minor amounts (up to 10%) of an alcohol, such as ethanol.

Preferably, the composition of the invention is a liquid formulation,including suspensions, aqueous solutions or in the form of an aerosol,all of which are known to those versed in the art. Aerosol formulationscan be placed into pressurized acceptable propellants, such as propane,nitrogen, and the like. They also may be formulated as pharmaceuticalsfor non-pressured preparations, such as in a nebulizer oratomizersuitable carriers.

DESCRIPTION OF THE SPECIFIC EXAMPLES

Influenza

Characterization of HN Antigen-Loaded Cationic Liposomes

Efficiency of encapsulation of HN (a commercial preparation ofhemagglutinin and neuraminidase derived from influenza viruses) loadedonto various cationic liposomal formulations, at different lipid/proteinw/w ratios (3/1-300/1), and with or without cholesterol (Chol) wastested. Table 1 shows the results of such an experiment, using thecationic lipids DOTAP and CCS. TABLE 1 The effect of the lipid (DOTAP,CCS)/protein ratio and cholesterol (Chol) on HN encapsulation efficiencyDOTAP/ DOTAP/ CCS/ HN Chol % HN CCS/ Chol % HN w/w mole encapsu- HN w/wmole encapsu- ratio ratio lation ratio ratio lation 300/1 1/1 93 300/11/0 73 100/1 1/1 90 100/1 1/0 64  50/1 1/1 90  30/1 1/0 38  30/1 1/1 88 10/1 1/0 1  10/1 1/1 79  3/1 1/1 35 100/1 1/0 90 300/1 3/2 71 100/1 1/192 100/1 3/2 64 100/1 2/1 89  30/1 3/2 41 100/1 4/1 80  10/1 3/2 0A monovalent vaccine was used for DOTAP and a trivalent vaccine for CCS.

The percentage of loading for DOTAP was 75-90% using a lipid/protein w/wratio of 50/1 to 300/1, with and without Chol. At lipid/protein w/wratios of 30/1 to 300/1, ˜90% antigen loading was achieved, decreasingto 79% and 35% at 10/1 and 3/1 w/w ratios, respectively. The addition ofChol to the formulation did not affect loading at DOTAP/Chol mole ratiosof 1/1 and 2/1, with slightly lower encapsulation (80%) at a ratio of4/1. For CCS, with or without Chol, the loading efficiency was lower(64-73% at w/w ratio of 100/1-300/1).

HN association with the liposomes upon simple mixing of the solubleantigen with preformed empty liposomes was also determined. In suchcases, 40-60% of the antigen was associated with the liposomes using alipid/protein w/w ratio of 100/to 300/1, regardless of the formulation.

These finding, collectively, indicate very high loading efficiency(>60%) using a simple and fast (5 min.) procedure in all formulations.Furthermore, even preformed liposomes in aqueous suspension were capableof effectively associating with the influenza virus surface antigens.

The immunogenicity of the various lipid/antigen w/w ratios (with orwithout the addition of cholesterol) was also evaluated.

In a first experiment sera levels of HI, IgG1 and IgG2a antibodiesfollowing i.n. vaccination of young (2-month-old) BALB/c mice withHN-loaded neutral, anionic or cationic liposomes were determined (Table2A). The HN antigen was a monovalent subunit vaccine derived from theA/New Caledonia (H1N1) strain. In the same experiment, lung and nasallevels of HI, IgG1, IgG2a and IgA antibodies, and INFγ levels producedby spleen cells, were also tested (Tables 2B and 2C). TABLE 2A Seralevels of HI, IgG1 and IgG2a antibodies Serum Group HI (n = 5) Vaccine(mean ± SD) IgG1 IgG2a 1 PBS  0 0 0 2 F-HN  0 55 0 3 Lip (DMPC)-HN(Neutral) 3 ± 7 150 0  (0) 4 Lip (DMPC/DMPG)-HN  6 ± 13 500 0 (Anionic) (20) 5 Lip (DC-Chol:DOPE)-HN 18 ± 7  0 0  (0) 6 Lip (DSTAP:Chol)-HN 28± 29 20 0  (40) 7 Lip (DDAB:Chol)-HN 136 ± 32  100 0 (100) 8 Lip(DOTAP:Chol)-HN 576 ± 128 15000 730 (100) 9 Lip (DMTAP:Chol)-HN 672 ±212 30000 470 (100) 10 Lip (CCS:Chol)-HN 2368 ± 1805 30000 9000 (100) 11F-HN + CT (1 μg) 1664 ± 572  55000 7000 (100)F-HN, free antigen; Chol, cholesterol; CT, cholera toxin. Groups 5-10are cationic liposomes. In parentheses, % seroconversion - % of micewith HI titer ≧ 40.

In particular, a comparison was made between neutral (DMPC), anionic(DMPC/DMPG, 9/1 mole ratio) and cationic (6 formulations) assemblies(Lip) encapsulating the HN antigens to induce local and systemicresponses following two i.n. administrations. For all formulations, thelipid/HN w/w ratio was 300/1, and the cationic lipid/Chol or cationiclipid/DOPE mole ratio was 1/1. Free antigen (F-HN) and F-HNco-administered with cholera toxin (CT, 1 μg) as an adjuvant were testedin parallel. The vaccine was given on days 0 and 7, 3 μg/dose (10 μl pernostril), and the responses were determined 46 weeks after the secondvaccine dose. TABLE 2B lung and nasal wash levels of IgG1, IgG2a and IgAantibodies Group Lung Nasal (n = 5) Vaccine IgG1 IgG2a IgA IgG1 IgG2aIgA 1 PBS 0 0 0 0 0 0 2 F-HN 0 0 0 0 0 0 3 Lip (DMPC)-HN (Neutral) 30 00 0 0 0 4 Lip (DMPC/DMPG)-HN 40 0 0 0 0 0 (Anionic) 5 Lip(DC-Chol:DOPE)-HN 0 0 0 0 0 0 6 Lip (DSTAP:Chol)-HN 0 20 0 0 80 0 7 Lip(DDAB:Chol)-HN 0 80 30 0 30 0 8 Lip (DOTAP:Chol)-HN 730 1050 170 40 18030 9 Lip (DMTAP:Chol)-HN 470 3000 30 15 80 70 10 Lip (CCS:Chol)-HN 900030000 1900 15000 30 300 11 F-HN + CT (1 μg) 7000 10000 1800 40 120 30

TABLE 2C Spleen INFγ levels (pg/ml) Group Spleen (n = 5) Vaccine IFNγ(pg/ml) 1 PBS 1800 2 F-HN 1400 3 Lip (DMPC)-HN (Neutral) 4200 4 Lip(DMPC/DMPG)-HN (Anionic) 4000 5 Lip (DC-Chol:DOPE)-HN 4900 6 Lip(DSTAP:Chol)-HN 2300 7 Lip (DDAB:Chol)-HN 3100 8 Lip (DOTAP:Chol)-HN8000 9 Lip (DMTAP:Chol)-HN 7800 10 Lip (CCS:Chol)-HN 10200 11 F-HN + CT(1 μg) 5200

As shown in Tables 2A-2C, the free antigen, as well as the neutral andanionic Lip-HN were virtually ineffective mucosal vaccines. In contrast,the cationic Lip-HN, particularly those designated DOTAP-HN, DMTAP-HNand CCS-HN evoked a robust systemic and mucosal humoral response, withhigh levels of IgG1, IgG2a and IgA antibodies, namely a mixed Th1+Th2response. No IgE antibodies were defected. The cationic liposomalvaccines comprising DOTAP-HN, DMTAP-HN and CCS-HN also induced highlevels of IFNγ (but not IL-4) in antigen-stimulated spleen cells. Theresponses produced by CCS-HN were even stronger than those induced byF-HN adjuvanted with CT. Based on these findings, only the cationicliposomal formulations: DOTAP-HN, DMTAP-HN and CCS-HN were further used.

In a second experiment, the effect of lipid/EN w/w ratio on theimmunogenecity of HN-loaded cationic liposomes and of preformedliposomes simply mixed with the soluble antigen, was determined. Thedata shown in Tables 3A-3C indicate that all three formulations induceda strong systemic (serum) and local (lung) response, and that loweringthe lipid/HN w/w ratio below 100/1 markedly reduced the response. TABLE3A Serum levels of HI, IgG1, IgG2a and IgA antibodies Lipid/HN No.Vaccine (n = 5) w/w ratio Serum HI IgG1 IgG2a IgA 1 F-HN 0 0 0 0 2 Lip(DOTAP)-HN 300/1 496 ± 295 (100) 15000 450 0 3 100/1 196 ± 119 (100)5000 280 0 4  30/1 36 ± 50 (80) 1000 200 0 5  10/1 28 ± 18 (60) 600 30 06  3/1 0 20 0 0 7 Lip (DMTAP)-HN 300/1 388 ± 260 (100) 2500 250 0 8100/1 208 ± 107 (100) 2200 600 0 9  50/1 130 ± 118 (80) 850 150 0 10 30/1 48 ± 71 (40) 450 0 0 11  10/1 24 ± 35 (40) 120 0 0 12 Lip (CCS)-HN300/1 560 ± 480 (100) 2000 1800 200 13 100/1 752 ± 504 (100) 6500 6000 014  50/1 272 ± 156 (100) 1900 700 0 15  30/1 112 ± 125 (80) 650 400 0 16 10/1 52 ± 68 (40) 275 440 0 17 F-HN + CT (1 μg) — 896 ± 350 (100) 300008000 120 18 F-HN + Lip (DOTAP) 300/1 864 ± 446 (100) 5000 1500 0 19F-HN + Lip (DMTAP) 300/1 320 ± 226 (100) 1900 400 0 20 F-HN + Lip (CCS)300/1 704 ± 525 (100) 30000 5000 500In groups 18-20 preformed liposomes were mixed with the soluble antigen.

TABLE 3B Lung levels of HI, IgG1, IgG2a and IgA Lipid/ HN Vaccine w/wLung No. (n = 5) ratio HI IgG1 IgG2a IgA 1 F-HN 0 0 0 0 2 Lip 300/1 40600 85 30 (DOTAP)-HN 3 100/1 40 500 20 0 4  30/1 30 250 35 0 5  10/1 20250 0 0 6  3/1 10 20 0 0 7 Lip 300/1 0 5500 200 1200 (DMTAP)-HN 8 100/10 7000 350 0 9  50/1 0 4500 250 0 10  30/1 0 1500 110 0 11  10/1 0 500 00 12 Lip 300/1 80 12500 3000 20000 (CCS)-HN 13 100/1 80 7000 5500 6500014  50/1 40 5500 900 20000 15  30/1 0 1500 200 0 16  10/1 0 500 200 0 17F-HN + CT — 80 45000 2250 3000 (1 μg) 18 F-HN + Lip 300/1 0 6000 5001200 (DOTAP) 19 F-HN + Lip 300/1 0 3750 225 1500 (DMTAP) 20 F-HN + Lip300/1 80 35000 3000 80000 (CCS)

TABLE 3C Spleen INFγ levels (pg/ml) Lipid/HN Spleen No. Vaccine (n = 5)w/w ratio IFNγ (pg/ml) 1 F-HN 7430 2 Lip (DOTAP)-HN 300/1 9780 3 100/142220 4  30/1 20440 5  10/1 20400 6  3/1 27780 7 Lip (DMTAP)-HN 300/1Not done 8 100/1 9  50/1 10  30/1 11  10/1 12 Lip (CCS)-HN 300/1 13100/1 14  50/1 15  30/1 16  10/1 17 F-HN + CT (1 μg) — 18 F-HN + Lip(DOTAP) 300/1 19 F-HN + Lip (DMTAP) 300/1 20 F-HN + Lip (CCS) 300/1

The superiority of Lip CCS-HN vaccine over the other vaccineformulations is again seen as reflected by the high levels of serum andlung IgG2a and IgA antibodies (groups 12-16). Interestingly, simplemixing of soluble antigen with preformed liposomes generated very potentvaccines (groups 18-20) that are equal to liposomes encapsulating theantigen. This suggests that real encapsulation of the antigen may not benecessary for the adjuvanticity of the cationic assemblies/liposomes.

In a further experiment the effect of cholesterol on the immunogenicityof the HN-loaded liposomes was tested. Tables 4A-4C show the results ofthis experiment, indicating that the addition of Chol slightly reducedthe systemic H[response to DOTAP-HN at 2/1 and 4/1 mole ratios (groups4, 5), but not at a 1/1 mole ratio (group 3), and moderately enhancesthe overall response to DMTAP-HN at all ratios (groups 7-9) and thelocal (lung) response CCS-HN at a 1/1 ratio (group 11). TABLE 4A Serumlevels of HI, IgG1, IgG2a and IgA antibodies Cat lipid/Chol No. Vaccine(n = 5) w/w ratio Serum HI IgG1 IgG2a IgA 1 F-HN — 0 0 0 0 2 Lip(DOTAP)-HN 1/0 320 ± 0 (100) 15000 450 0 3 Lip (DOTAP:Chol)-HN 1/1 496 ±295 (100) 15000 450 0 4 2/1 168 ± 216 (100) 7000 800 0 5 4/1 195 ± 111(100) 15000 250 0 6 Lip (DMTAP)-HN 1/0 320 ± 188 (100) 20000 290 0 7 Lip(DMTAP:Chol)-HN 1/1 672 ± 419 (100) 30000 300 0 8 2/1 576 ± 368 (100)25000 650 0 9 4/1 608 ± 382 (100) 30000 600 0 10 Lip (CCS)-HN 1/0 2560 ±1568 (100) 30000 7000 100 11 Lip (CCS:Chol)-HN 1/1 2368 ± 1805 (100)30000 9000 100 12 F-HN + CT (1 μg) — 1664 ± 572 (100) 55000 7000 20

TABLE 4B Lung levels of HI, IgG1, IgG2a and IgA antibodies Cat lipid/Chol No. Vaccine w/w Lung No. (n = 5) ratio HI IgG1 IgG2a IgA 1 F-HN — 00 0 0 2 Lip 1/0 40 900 85 25 (DOTAP)-HN 3 Lip 1/1 40 600 80 30(DOTAP:Chol)-HN 4 2/1 40 680 180 22 5 4/1 60 720 50 60 6 Lip 1/0 60 100040 0 (DMTAP)-HN 7 Lip 1/1 120 3000 30 15 (DMTAP:Chol)-HN 8 2/1 160 2500160 200 9 4/1 80 4000 100 150 10 Lip 1/0 640 30000 1500 9000 (CCS)-HN 11Lip 3/2 1280 30000 1900 15000 (CCS:Chol)-HN 12 F-HN + CT — 20 10000 18001000 (1 μg)

TABLE 4C Spleen INFγ levels (pg/ml) No. Vaccine Cat lipid/Chol SpleenNo. (n = 5) w/w ratio IFNγ (pg/ml) 1 F-HN — 7430 2 Lip (DOTAP)-HN 1/07480 3 Lip (DOTAP:Chol)-HN 1/1 9780 4 2/1 12870 5 4/1 9330 6 Lip(DMTAP)-HN 1/0 8520 7 Lip (DMTAP:Chol)-HN 1/1 10900 8 2/1 8560 9 4/17490 10 Lip (CCS)-HN 1/0 15550 11 Lip (CCS:Chol)-HN 3/2 13780 12 F-HN +CT (1 μg) — 11110

The immunogenicity of CCS-HN vaccine was also evaluated in aged (18month) C57BL/6 mice following intramuscular (once on day 0) orintranasal (twice, days 0 and 7) administration of 1 μg and 2 μg,respectively, of subunit (HN) vaccine (derived from A/Panama [H3N2]virus). The lipid assemblies were composed of CCS/cholesterol (3:2 molarratio) and the lipid/HN w/w ratio was 200:1. As opposed to zero activityof the commercial vaccine, the CCS-HN vaccine evoked high levels ofserum HI and IgG2a antibodies (tested at 4 weeks post vaccination) andlung (tested at 6 weeks post vaccination) IgG2a and IgA antibodies ascan be seen in Tables 5A and 5B (the data show mean titers). TABLE 5ASerum levels of HI, IgG1, IgG2a and IgA in aged mice No. Vaccine^(a) (n= 5) Serum HI IgG1 IgG2a 1 PBS i.n. × 2 0 0 0 2 F-HN i.m. × 1 0 15 0 3F-HN i.n. × 2 0 0 0 4 Lip (CCS)-HN i.n. × 2 80 130 350

TABLE 5B Lung levels of IgG1, IgG2a and IgA in aged mice No. Vaccine (n= 5) Lung IgG1 IgG2a IgA 1 PBS i.n. × 2 0 0 0 2 F-HN i.m. × 1 0 0 0 3F-HN i.n. × 2 0 0 0 4 CCS-HN i.n. × 2 0 180 840

In addition, the induction of cellular responses by the various vaccineformulations was tested. In particular, young mice were immunized i.n.with various cationic liposomal formulations and the splenocyte cellularresponses—cytotoxicity, proliferation and IFNγ production—were measured6 weeks after vaccination. In the experiment, the results of which areshown in Table 6, a comparison was made between HN-loaded liposomes(groups 3-10) and free antigen (F-HN) given alone (group 2) or admixedwith preformed empty liposomes (groups 11-13). The immunogenicity of Lip(DMTAP)-HN and Lip (CCS)-HN prepared at varying lipid/HN w/w ratios(30/1-300/1) was also determined. TABLE 6 Induction of cellularresponses by cationic liposomes administered i.n. Lipid/ Prolif- HN %cytotoxicity eration w/w P815 + Δcpm IFNγ No. Vaccine ratio peptide P815(mean) (pg/ml) 1 PBS — 6 4 7010 1900 2 F-HN — 8 5 7700 4500 3 Lip(DMTAP)-HN 300/1 16 13 10960 3500 4 100/1 9 9 12870 5850 5  50/1 3 217670 3400 6  30/1 3 2 17920 3050 7 Lip (CCS)-HN 300/1 4 2 20370 8000 8100/1 21 7 24870 8250 9  50/1 6 3 20980 10650 10  30/1 8 5 11510 3500 11F-HN + Lip 300/1 17 4 19390 3400 (DOTAP) 12 F-HN + Lip 300/1 17 7 118505700 (DMTAP) 13 F-HN + Lip 300/1 16 8 19270 4100 (CCS)

Preferential cytotoxicity against the specific target cells (P815 pulsedwith the influenza peptide) was obtained only with CCS-HN at a lipid/HNw/w ratio of 100/1 (group 8) and with all the three preformed liposomes(DOTAP, DMTAP and CCS) co-administered with free antigen. The maximumproliferative response was observed with DMTAP-HN at lipid/HN w/w ratiosof 50/1 and 30/1 and with CCS-HN at 300/1, 100/1 and 50/1 ratios. Theproliferative and cytotoxic responses elicited by the most efficaciousliposomal formulations were 2-3 times greater than those induced by freeantigen.

These findings suggest that as compared with the humoral response (Table3), where the highest levels of all types of antibodies measured wereobtained at lipid/HN w/w ratios of 100/1-300/1, lower w/w ratios (e.g.30/1-100/1) may be optimal for the cellular responses. Moreover, whereasDMTAP-HN elicits a strong humoral response, this formulation is a poorinducer of cytotoxic activity, as compared with CCS-HN. Interestingly,vaccination with mixtures of free antigen with preformed cationicliposomes (all three formulations) in suspension evokes good cellularresponses that are similar in magnitude to those induced by theencapsulated antigen. Thus, simple mixing of free antigen with preformedcationic liposomes may be sufficient to induce both strong humoral(Table 3A-3C) and cellular (Table 6) responses.

In yet a further experiment, the results of which are shown in Tables7A-7C, a comparison was made between 1 i.m. dose, 1 or 2 i.n. doses and2 oral doses of a monovalent HN-loaded cationic liposomes comprisingDOTAP, DMTAP or CCS with regard to immunogenicity and induction ofprotective immunity to live virus challenge. In this experiment, thelipid/N w/w ratio was 300/1 and the cationic lipid/Chol ratio was 1/1for DOTAP and DMTAP systems and 3/2 for CCS system. Of the three routes,i.n. administration twice generates the strongest humoral and cellularresponse and protective immunity. Of the 3 formulations, CCS induces thehighest response, particularly with regard to IgG2a and IgA antibodies.TABLE 7A Serum levels of HI, IgG1, IgG2a and IgA Serum No. Vaccine (n =10) Route HI IgG1 IgG2a IgA 1 PBS 0 0 0 0 2 F-HN i.m. × 1 60 ± 37 (70)1000 40 0 3 oral × 2 0 0 0 0 4 i.n. × 1 0 0 0 0 5 i.n. × 2 0 55 0 0 6Lip (DOTAP/Chol)-HN i.m. × 1 424 ± 141 (100) 21000 5500 0 7 oral × 2 0 00 0 8 i.n. × 1 40 ± 28 (50) 450 80 0 9 i.n. × 2 409 ± 172 (100) 250001300 60 10 Lip (DMTAP/Chol)-HN i.m. × 1 768 ± 211 (100) 24000 8000 0 11oral × 2 0 0 0 0 12 i.n. × 1 10 ± 10 (0) 300 60 0 13 i.n. × 2 532 ± 763(100) 10500 380 50 14 Lip (CCS/Chol)-HN i.m. × 1 864 ± 1100 (100) 2500010000 0 15 oral × 2 0 0 16 i.n. × 1 34 ± 50 (20) 1000 30 0 17 i.n. × 22289 ± 1576 (100) 25000 20000 400 18 F-HN + CT (1 μg) i.n. × 2 756 ± 650(100) 21000 15000 20

TABLE 7B Lung antibodies Vaccine Lung No. (n = 5) Route HI IgG1 IgG2aIgA 1 PBS 0 0 0 0 2 F-HN i.m. × 1 0 80 0 0 3 oral × 2 0 0 0 0 4 i.n. × 10 0 0 0 5 i.n. × 2 0 70 20 0 6 Lip (DOTAP/ i.m. × 1 40 900 500 0Chol)-HN 7 oral × 2 0 0 0 0 8 i.n. × 1 0 50 20 0 9 i.n. × 2 120 100001000 350 10 Lip (DMTAP/ i.m. × 1 20 900 150 0 Chol)-HN 11 oral × 2 0 0 00 12 i.n. × 1 0 35 20 0 13 i.n. × 2 240 20000 700 2200 14 Lip (CCS/ i.m.× 1 60 3500 900 0 Chol)-HN 15 oral × 2 0 0 0 0 16 i.n. × 1 0 120 0 35 17i.n. × 2 360 30000 5000 20000 18 F-HN + CT i.n. × 2 240 22000 2500 1800(1 μg)

TABLE 7C Cellular response and protective immunity Spleen Lung VaccineΔcpm IFNγ Virus titer No. (n = 5) Route (mean) (pg/ml) (log 10) 1 PBS1641 0 7 2 F-HN i.m. × 1 1909 0 4 3 oral × 2 2253 0 ND 4 i.n. × 1 669 0ND 5 i.n. × 2 2813 0 5 6 Lip (DOTAP/ i.m. × 1 3452 3300 0 Chol)-HN 7oral × 2 0 1150 ND 8 i.n. × 1 482 1900 ND 9 i.n. × 2 8391 3200 0 10 Lip(DMTAP/ i.m. × 1 5632 0 1 Chol)-HN 11 oral × 2 553 0 ND 12 i.n. × 1 12770 ND 13 i.n. × 2 7331 3150 0 14 Lip (CCS/ i.m. × 1 6196 5750 0 Chol)-HN15 oral × 2 476 550 ND 16 i.n. × 1 1705 6250 ND 17 i.n. × 2 4912 15500 018 F-HN + CT i.n. × 2 1933 5650 0 (1 μg)

In the experiment described in Tables 8-10, a commercial trivalentvaccine was tested and a comparison was made between a single CCS-basedvaccine dose (using 2 or 4 μg of antigen [HN] of each viral strain) andtwo vaccine doses (2 μg/strain/dose), given at 3, 7 or 14 day intervalsbetween administrations. The lipid assemblies were composed of CCS/Chol(cholesterol) at a 3/2 mole ratio, and the lipid/HN w/w ratio was 100/1for all formulations. As controls, the standard trivalent commercialvaccine (ES) was administered either alone or combined with 1 μg choleratoxin (CT), used as a mucosal adjuvant. Sera, lung homogenates and nasalwashes were tested 5-6 weeks after the first vaccine dose for HIantibodies (Table 8), as well as for antigen-specific IgG1, IgG2a, IgAand IgE antibodies (Table 9). In addition, 5 mice from selected groupswere challenged i.n. with live virus (using the mouse adaptedreassortant X-127 virus) and protection was assessed by quantifying lungvirus titer 4 days later (Table 10).

As opposed to the poor or no immunogenicity of the commercial fluvaccine (HN) (groups 2-6), CCS/Chol-flu vaccine induced high titers ofall types of antibodies tested (except for IgE which was undetected),especially against the two A virus strains (groups 8-11; Tables 8, 9).For the 2-dose regimen, a 1-week interval appears to be the optimal (gr.10). For the single dose regimen, 4 μg antigen, but not 2 μg (gr. 8 vs.gr. 7), induced high titers of serum HI, IgG1 and IgG2a antibodies andlung IgG1 antibodies. However, in comparison with the 2-dose regimen,the 1-dose regimen did not elicit lung IgG2a and IgA antibodies nornasal antibodies (Table 9).

In the protection assay (Table 10), the CCS-flu vaccine administeredi.n. either once (4 μg) or twice (2 μg/dose) afforded full protectionagainst viral infection (6 log reduction in lung virus titer) whereasthe standard vaccine reduced virus titer by only 0.5-1 log. Thus,although the single dose regimen with the CCS-flu vaccine is inferior tothe two-dose regimen for certain antibody isotypes, the two regimensprovide a similar degree of protection.

In this experiment, we also compared CCS alone to CCS/Chol as thevaccine carrier, and found no difference in immunogenicity between thetwo formulations (data not shown). Another formulation modification wasthe reduction of the size of the CCS/Chol lipid assemblies (diameter0.05-5 μm) by extrusion (diameter≦0.02 μm). Antibody titers induced bythe extruded vaccine were 50-80% lower than those produced by thenon-extruded vaccine (data not shown). Thus, unsized CCS lipidassemblies, with or without cholesterol, are highly efficient as avaccine carrier for trivalent flu vaccine. TABLE 8 Elicitation ofhemagglutination inhibition (HI) antibodies following intranasalvaccination with trivalent influenza vaccine, free and in CCS lipidassemblies, administered once or twice at various time intervals toyoung (2 mo.) BALB/C mice Mean HI titer (% seroconversion)^(b) DosingA/New Caledonia A/Panama B/Yamanashi No. Vaccine^(a) (n = 5) days serumlung serum lung serum lung 1 None (PBS) ×2 0, 7 0 0 0 0 0 0 2 F-HN 2 μg× 1 0 0 0 0 0 0 0 3 4 μg × 1 0 0 0 0 0 0 0 4 2 μg × 2 0, 3 0 0 0 0 0 0 52 μg × 2 0, 7 0 0 0 0 0 0 6 2 μg × 2 0, 14 0 0 0 0 0 0 7 Lip(CCS/Chol)-HN 2 μg × 1 0 0 0 0 0 0 0 8 4 μg × 1 0 336 (100) 40 328 (100)40 52 (80) 0 9 2 μg × 2 0, 3 544 (100) 80 408 (100) 40 52 (80) 0 10 2 μg× 2 0, 7 544 (100) 80 544 (100) 120  88 (100) 0 11 2 μg × 2 0, 14 480(100) 60 368 (100) 40 80 (80) 0 12 F-HN + CT (1 μg) 2 μg × 2 0, 7 608(100) 80 664 (100) 120 84 (80) 0^(a)Mice were immunized with Fluvirin ® 2003/2004 trivalent subunitvaccine preparation consisting of A/New Caledonia/20/99 (H1N1)-like,A/Moscow/10/99 (H3N2)-like and B/Hong Kong/330/2001-like, either free(F-HN) or incorporated into CCS/Chol (3/2 mole ratio) lipid assemblies(0.6 mg for groups 7, 9, 10, 11; 1.2 mg for group 8).^(b)Serum HI titer was determined on individual mice 35 days after thefirst vaccine dose. Lung (pooled) HI titer was tested on day 42.In parentheses - % of mice with HI titer ≧40. 0 denotes HI titer <20.

TABLE 9 Elicitation of serum, lung and nasal antigen-specific IgG1,IgG2a and IgA antibodies following intranasal vaccination with trivalentinfluenza vaccine, free and in CCS lipid assemblies, administered onceor twice at various intervals to young (2 mo.) BALB/c mice Mean antibodytiter Dosing Serum Lung Homogenate Nasal wash No. Vaccine^(a) (n = 5)days IgG1 IgG2a IgG1 IgG2a IgA IgG1 IgG2a IgA 1 None (PBS) ×2 0, 7 0 0 00 0 0 0 0 2 F-HN 2 μg × 1 0 0 0 0 0 0 0 0 0 3 4 μg × 1 0 320 90 1500 0 00 0 0 4 2 μg × 2 0, 3 0 0 0 0 0 0 0 0 5 2 μg × 2 0, 7 0 0 0 0 0 0 0 0 62 μg × 2 0, 14 40 0 0 0 0 0 0 0 7 Lip (CCS/Chol)-HN 2 μg × 1 0 300 0 6000 0 0 0 0 8 4 μg × 1 0 12000 4500 13000 0 0 0 0 0 9 2 μg × 2 0, 3 1500010000 15000 2500 3500 0 10 0 10 2 μg × 2 0, 7 15000 12000 14000 25009000 200 30 100 11 2 μg × 2 0, 14 13000 5500 12000 1800 3000 50 0 0 12F-HN + CT (1 μg) 2 μg × 2 0, 7 21000 15000 20000 2500 2000 250 30 45^(a)See table 8 for experimental details. Samples were pooled and testedby ELISA against the 3 viral strains (pooled HN) 42 days after the firstvaccine dose. 0 denotes titer <10.

TABLE 10 Protection of young BALB/c mice against viral challengefollowing intranasal vaccination with trivalent influenza vaccine, freeand in CCS lipid assemblies Dosing Lung virus No. Vaccine^(a) (n = 5)days titer (log 10)^(b) 1 None — 6 2 F-HN 4 μg × 1 0 5.5 3 F-HN 2 μg × 20, 7 5 4 Lip (CCS/Chol)-HN 4 μg × 1 0 0 5 Lip (CCS/Chol)-HN 2 μg × 2 0,7 0 6 F-HN 2 μg + CT (1 μg) × 2 0, 7 0^(a)See table 8 for experimental details. In groups 4, 5 the lipid/HNw/w ratio was 100/1.^(b)The mice were infected intranasally 42 days after the first vaccinedose, using ˜10⁶ egg infectious dose 50% (EID 50) of the mouse-adaptedreassortant X-127 virus (A/Beijing/262/95 [H1N1] × X-31 [A/HongKong/1/68 × A/PR/8/34).# Lungs were harvested 4 days later, homogenized, serially diluted, andinjected into the allantoic sac of 10 d. fertilized chicken eggs. After48 h at 37° C. and 16 h at 4° C., 0.1 mL of allantoic fluid was removedand checked for viral presence by hemagglutination.

In the experiment described in Tables 11 and 12, the trivalent-fluvaccine was formulated with the CCS/Chol lipid assemblies using varyingamounts of the HN antigens and the lipid. In this experiment thevaccines were prepared with: (a) varying amounts of the antigen (0.25-2μg per viral strain) and of the lipid (0.075-0.6 mg), keeping thelipid/HN w/w ratio constant at 100/1; (b) graded amounts of the antigen(0.25-2 μg) and a constant amount of the lipid (0.6 mg) thereby varyingthe lipid/HN w/w ratio from 100/1 to 800/1. As can be seen in Table 11(HI titer) and Table 12 (isotype titers) vaccines prepared at a 100/1lipid/HN w/w ratio using 2 or 1 μg antigen of each strain and 0.6 or 0.3mg lipid, respectively, produced high and similar levels of antibodiesagainst the 3 viral strains (groups 2, 3). At lower antigen (0.5, 0.25μg/strain) and lipid (0.15, 0.075 mg) doses the response decreasedmarkedly (groups 4, 5), particularly the mucosal response (lung, nasal)(Table 12). When a constant dose of lipid was used (0.6 mg), high levelsof antibodies were obtained even with the two lower doses of antigen(0.25, 0.5 μg/strain) (groups 6-8). Thus, the amount of the CCS lipid iscritical, and with the appropriate lipid dose the antigen dose can bereduced 4-8 fold (from 1-2 μg to 0.25-0.5 μg). TABLE 11 Effect of theantigen dose and lipid dose on the induction of HI antibodies followingintranasal vaccination with trivalent influenza vaccine formulated withCCS lipid assemblies, administered twice (at 1 week interval) to young(2 mo.) BALB/c mice Mean HI titer (% seroconversion) HN Lipid Lipid/HNA/New Caledonia A/Panama B/Yamanashi No. Vaccine^(a) (n = 5) (μg) (mg)w/w ratio Serum Lung Serum Lung Serum Lung 1 F-HN 2 — — 0 0 0 0 0 0 2Lip (CCS/Chol)-HN 2 0.6 100/1 544 (100) 80 544 (100) 120 88 (100) 0 3 10.3 100/1 320 (100) 80 544 (100) 160 40 (100) 0 4 0.5 0.15 100/1 416(100) 20 448 (100) 40 32 (100) 0 5 0.25 0.075 100/1 180 (100) 0 100(100) 20 0 0 6 1 0.6 200/1 672 (100) 80 736 (100) 160 104 (100)  0 7 0.50.6 400/1 560 (100) 80 608 (100  160 104 (100)  0 8 0.25 0.6 800/1 512(100) 80 512 (100) 120 48 (100) 0^(a)See Table 8 for experimental details.

TABLE 12 Effect of the antigen dose and lipid dose on the induction ofserum, lung and nasal antigen-specific IgG1, IgG2a and IgA antibodiesfollowing intranasal vaccination with trivalent influenza vaccineformulated with CCS lipid assemblies, administered twice (at 1 weekinterval) to young BALB/c mice Mean antibody titer HN Lipid Lipid/HNSerum Lung homogenate Nasal wash Vaccine^(a) (n = 5) (μg) (mg) w/w ratioIgG1 IgG2a IgG1 IgG2a IgA IgG1 IgG2a IgA 1 F-HN 2 — — 0 0 0 0 0 0 0 0 2Lip (CCS/Chol)-HN 2 0.6 100/1 15000 12000 14000 2500 9000 200 30 100 3 10.3 100/1 14000 2500 10000 1000 8000 100 0 80 4 0.5 0.15 100/1 150001300 8000 1500 4000 0 0 0 5 0.25 0.075 100/1 12000 400 3500 400 2500 0 00 6 1 0.6 200/1 20000 15000 12000 2500 8000 200 15 80 7 0.5 0.6 400/115000 14000 15000 5000 15000 150 35 100 8 0.25 0.6 800/1 15000 900021000 2500 13000 250 25 90^(a)See Tables 8, 9 for experimental details.

In a further experiment, the subunit flu vaccine, either free (HN) orassociated with the CCS/Chol lipid assemblies (Lip HN), was tested forits ability to induce HI antibodies cross-reacting with variousinfluenza A and B substrains that were not included in the vaccine. Thedata shown in Table 13 indicate that intranasal (i.n.) and intramuscular(i.m.) vaccination, administered once or twice, with either a monovalentor trivalent CCS-based influenza vaccine elicits high serum titers of HIantibodies directed against the immunizing strains, as well as HIantibodies cross-reacting with several A/H1N1, A/H3N2 and B strains thatwere circulating in the years 1986-1999 and were not included in thevaccine. Slightly lower HI titer were found after a single i.n. vaccinedose (gr. 6 vs. gr. 7). Lung homogenate HI titers (gr. 4, 8) were lowerthan the corresponding serum titers. Thus, parenteral or intranasalvaccination with the CCS-based vaccine may afford protection against awide spectrum of A and B viral strains. Such antigenic variants mayemerge during a flu epidemic/pandemic as a result of antigenic drift. Incontrast, the standard commercial vaccine administered i.n. (gr. 1, 5)was totally ineffective in inducing antibodies against both thehomologous and the heterologous strains. TABLE 13 Induction of straincross-reactive HI antibodies following intranasal or intramuscularvaccination of young BALB/c mice with CCS-based monovalent and trivalentinfluenza vaccine Mean HI titer against: A/H1N1 New A/H3N2 B Cale-Singa- Nan- Johannes- Yama- Vaccine Sample donia/ Beijing/ Texas/ pore/Panama/ Sydney/ chang/ burg/ nashi/ Harbin/ No. Vaccine^(a) strainstested 20/99 262/95 36/91 6/86 2007/99 5/97 333/95 33/94 166/98 07/94 1HN A/New serum 0 0 0 0 0 0 0 0 0 0 2 μg × 2 i.n. Caledonia 2 Lip HNserum 1280 1280 1280 240 0 0 0 0 0 0 2 μg × 2 i.n. 3 Lip HN serum 640640 320 40 0 0 0 0 0 0 1 μg × 1 i.m. 4 Lip HN lung 320 240 240 20 0 0 00 0 0 2 μg × 2 i.n. homogenate 5 HN A/New serum 0 0 0 0 0 0 0 0 0 0 2 μg× 2 i.n. Caledonia, 6 Lip HN A/Panama, serum 320 80 120 0 320 320 120120 60 120 4 μg × 1 i.n. B/Hong 7 Lip HN Kong serum 480 120 240 20 640640 120 120 80 320 2 μg × 2 i.n. 8 Lip HN lung 80 80 40 0 120 80 0 0 040 2 μg × 2 i.n. homogenate 9 HN serum 480 240 120 40 480 480 120 120 80240 2 μg + CT 1 μg × 2 i.n.^(a)Pooled sera and lung homogenate obtained 5 weeks after vaccinationwere tested for HI antibodies. For experimental details, see Table 8.The lipid (Lip) assemblies were composed of CCS/Chol (3/2 mole ratio)and the lipid/HN w/w ratio was 300/1 in groups 2-4 and 100/1 in groups6-8. Except for groups 3 and 6, the two vaccine doses were spaced 1 weekapart. In bold, antibody titers against the immunizing strains. 0denotes HI titer <10.Biodistribution of Anionic and Cationic Liposomes Loaded with HN andAdministered Intranasally

In a biodistribution experiment, 3 formulations of lipid assemblies:DMPC/DMPG (anionic), DOTAP/Chol (cationic) and CCS/Chol (cationic),either empty or loaded with the influenza HN antigens, were administeredintranasally (200 μg lipid, 2 μg antigen per mouse) into BALB/c mice.The fluorescently labeled lipid was then traced in the homogenates ofvarious tissues over a period of 24 h (at 1, 5, and 24 hours postadministration).

As can be seen in the following Table 14 and in FIG. 2A-2F, after 1 and5 hours there was 75-100% recovery of the administered lipid of all thethree formulations tested. This recovery however dropped significantlyat 24 hours in all formulations except for the CCS formulation. The CCSformulation containing the HN antigens displayed the longest retention(>24 h.) in the 3 target organs (nose, lungs, GI tract) while there wasno lipid accumulation in the brain and no significant accumulation inthe other organs tested (liver, kidneys, heart, spleen). TABLE 14Recovery at 1, 5, and 24 hours of fluorescently labeled lipid assembliesadministered intranasal % Recovery (of total lipid administered) Lipidassembly formulation 1 hour 5 hours 24 hours DMPC/DMPG (empty) 100.299.3 26.9 DMPC/DMPG:HN 100.2 99.9 8.3 DOTAP/Chol (empty) 107.0 75.1 8.1DOTAP/Chol:HN 99.9 106.4 6.7 CCS/Chol (empty) 99.6 96.9 74.2 CCS/Chol:HN101.1 101.5 94.5

When ¹²⁵I-labeled HN was used, its biodistribution resembled that of thefluorescent lipid (data not shown). This long retention of the CCSvaccine components in the respiratory and GI tracts may explain, inpart, its superior immunogenicity over the other liposomal formulations.This is exhibited in the following study in which the antigen componentof the vaccine was traced. HN proteins were labeled with ¹²⁵I andadministered intranasally either free or associated with one of thelipid formulations used in the fluorescent biodistribution experiments.Radioactivity of the various tissues was determined at 1, 5 and 24 hpost instillation.

Table 15 teaches that recovery of the antigen was high in thisexperiment as well. As can be seen in FIG. 3A-3D, the biodistributionpattern of the ¹²⁵I-labeled HN is similar to that of the lipid (FIG.2A-2F), further establishing that: (a) there is indeed an in vivoassociation between the HN-proteins and the lipid assemblies, and (b)the prolonged retention in the nose of the antigen when associated withthe cationic lipid assemblies may be due to the cationic lipidassemblies and not an inherent property of the HN proteins, since thereis no HN retention when the protein is administered by itself in solubleform.

Also this experiment may teach that there is no HN protein accumulationin the brain when administered alone or associated with lipid-assemblies(a major safety concern with intranasal vaccination). Since theradioactive tracing method is much more sensitive than the fluorescentmethod, this result is more confidently based. TABLE 15 Recovery of ¹²⁵Ilabeled HN administered intranasally either alone or associated withlipid assemblies at 1, 5 and 24 hours Recovery (% of total injected)Lipid assembly formulation 1 hour 5 hours 24 hours HN 77 48 17 Lip(DMPC:DMPG) HN 88 50 26 Lip (DOTAP:Chol) HN 105 58 32 Lip (CCS:Chol) HN100 74 41

In an attempt to test if the protein and lipid are retained and/orcleared by similar or different kinetics in the various tissues, anotheranalysis of the data was performed, where the ratio between the %antigen retention (of the total dose administered) and % lipid retentionin the various tissues at various time points was determined. When theratio is constant and =1, it means that both components were similarlyretained in the same organ, while when this ratio is either larger orsmaller than 1 it suggests that the clearance kinetics of each componentwas different, and one component was cleared faster than the other.

As can be seen in Table 16 below, the only ratio that remained constantwith time was that of CCS/Chol-HN in the nose (ratio=˜0.45). Thissuggests that: (a) the high retention of the antigen in the nose withCCS and DOTAP is in correlation with the level of association and due tothe binding of these formulations to the nasal mucosa, in contrast toDMPC/DMPG; and (b) while the other formulations' components dissociatein the body and are cleared at different rates, the CCS-HN basedformulation was stable, especially in the nose, and this may contributeto the enhanced immunogenicity seen with the CCS-based vaccines. TABLE16 Retention of the lipid and HN antigen after i.n. administration Lip(DMPC:DMPG) HN Lip (DOTAP:Chol) HN Lip (CCS:Chol) HN 1 h 5 h 24 h 1 h 5h 24 h 1 h 5 h 24 h HN nose  9%  4% 2% 38% 16% 2% 41% 14% 12% lungs 30% 3% 4% 19%  4% 12%  24% 21% 11% GI 35% 32% 11%  33% 27% 10%  22% 28%  8%recovery: 88% 50% 26%  105%  58% 32%  100%  74% 41% lipid nose  0%  0%0% 46% 56% 0% 88% 30% 25% lungs 67% 80% 5% 38%  3% 3% 12% 35% 14% GI 33%20% 4% 16% 47% 3%  1% 37% 55% recovery: 100%  100%  8% 100%  106%  7%101%  102%  95% HN/lipid nose — — — 0.82 0.29 — 0.47 0.45 0.47 ratiolungs 0.44 0.03 0.85 0.50 1.29 3.56 2.01 0.60 0.80 GI 1.07 1.56 2.962.12 0.57 2.86 16.08  0.76 0.15 recovery: 0.88 0.50 3.12 1.05 0.55 4.780.99 0.73 0.44The values show % retention of the total lipid or HN proteinadministered.Preliminary Safety Study of the Intranasal Flu Vaccine

Toxicity (local, systemic) is a major concern with both i.m. and i.n.vaccines and therefore a pilot toxicity study was studied. Cationiclipid formulations (DMTAP, DOTAP, CCS-based) loaded with the influenzaantigens hemagglutinin+neuraminidase (HN) were administered i.n. (twice,spaced 1 week apart) to mice (n=4/group), and blood counts (total,differential), blood chemistry and histological examination (nose, lungsections) were performed 72 hours later. The mice showed no apparentsigns of any toxicity. Blood counts and blood chemistry were within thenormal range, and, as expected, minimal-mild inflammatory response wasseen in the nose and lungs of mice treated with the cationicformulations. A similar, albeit less pronounced, inflammatory responsewas also seen in some mice treated with saline alone or with thenon-encapsulated antigen.

Immunomodulatory Activity of CCS-Flu Vaccine in Mice

In these experiments, mice were injected i.p. with various liposomalformulations (composed of DMPC, DMPC/DMPG, DOTAP/Chol, CCS/Chol), 0.5-1mg lipid, with or without the HN antigens. The mice were eitheruntreated or i.p. injected with thioglycollate (TG, to increasemacrophage production) 2 days before the injection of the liposomalformulations. Peritoneal cells were harvested 24-48 h. afteradministration of the liposomes and used as such or after 4 h.adsorption at 37° C. to plastic dishes and removal of the non-adherentcells. In other experiments, peritoneal cells were harvested from TGtreated mice and incubated with the liposomal formulations for 24-48 h.The cells were tested by flow cytometery for the expression of MHC IIand the co-stimulatory molecules CD40 and B7. The supernatants weretested for the cytokines interferon γ (IFN γ), tumor necrosis factor α(TNF α) and interleukin 12 (IL-12), and for nitric oxide (NO).

All the cationic formulations (CCS/Chol, DOTAP/Chol, DMTAP/Chol)upregulated the expression of B7 and CD40 more than the otherformulations (DMPC [neutral], DMPC/DMPG [anionic]) and induced higherlevels of IFN γ and IL-12. In some cases the CCS/Chol formulation wasmore effective than the other cationic formulations. No significantlevels of TNF α and NO were induced by any of the formulations. Theenhanced expression of co-stimulatory molecules on antigen presentingcells and the induction of IL-12 and IFNγ by the cationic formulationscan explain, in part, the greater adjuvant activity of theseformulations. These findings combined with the long retention of theCCS-flu vaccine in the respiratory tract (FIGS. 2C and 2F and FIG.3A-3D) after intranasal administration may explain why CCS is such anefficient mucosal vaccine carrier/adjuvant.

Hepatitis A Virus (HAV)

In addition to influenza, the immune enhancing potential of CCS lipidassemblies was also tested for HAV vaccine administered by theintranasal (i.n.) and the intrarectal (i.r.) routes.

HAV vaccine (Aventis Pasteur), 10 EU (˜1.5 μg protein), was administeredtwice at a 2-week interval and the response was tested by the ELISPOTtechnique 3 weeks after the second vaccine dose. CpG-ODN, used as amucosal adjuvant, was given at 10 μg/dose. The HAV-CCS lipid assemblieswere prepared as described above for the influenza vaccine (Table 1).

The data presented in Table 17 show that whereas the commercial HAVvaccine failed to induce an IgA response in both tissues (laminapropria, Peyer's patches) tested, and by both administration routes(i.n., i.r.), the vaccine formulated with either CCS or CpG-ODNgenerated a significant response in most cases. The combination ofHAV-CCS lipid assemblies and CpG-ODN resulted in a synergistic responsein all cases. Thus, CCS lipid assemblies alone, and particularly incombination with CpG-ODN, are also effective as a carrier/adjuvant formucosal vaccination against HAV. TABLE 17 Induction of IgA antibodiesfollowing intranasal (i.n.) or intrarectal (i.r.) vaccination of BALB/cmice with hepatitis A virus (HAV) vaccine, alone and in combination withCCS lipid assemblies and/or CpG-ODN Mean no. of IgA AFC/10⁶ cells in:Lamina propria Peyer's patches Vaccine i.n. i.r. i.n. i.r. HAV alone 0 00 0 HAV-CCS 12 27 0 1 HAV + CpG-ODN 16 22 0 14 HAV-CCS + CpG-ODN 139 6828 23AFC—antibody-forming-cells

C. Botulinum

In a further experiment, Mice were immunized i.n. with 0.4 μg dose of acommercial C. botulinum toxoid (as a model for bioterror agent, Uruguay,alum free) and antibody titers were tested by ELISA 4 weeks after thesecond vaccine dose.

The results of an experiment with C. botulinum toxoid are summarized inTable 18, which shows the superiority of the CCS-toxoid formulation overthe standard vaccine following i.n. instillation, particularly withregard to the IgA levels in the small intestine and feces. Such Abs areexpected to neutralize the toxin upon oral exposure. Mice immunized i.n.with the vaccine alone did not produce IgA. TABLE 18 Induction of IgG1,IgG2a and IgA antibodies in BALB/c mice vaccinated intranasally (twice,1 week apart) with free or CCS-associated Clostridium botulinum toxoid(CBT) Mean antibody titer Vaccine^(a) Serum Small intestine Feces n = 10IgG1 IgG2a IgG1 IgG2a IgA IgA CBT 0 0 1000 180 0 0 CCS-CBT 400 24 1600 01800 1800MaterialsChemistrySynthesis of N-palmitol D-erthro sphingosyl-1-carbamoyl Spermine (CCS)

(i) N-palmitoylsphingosine (1.61 g, 3 mmol) was dissolved in dry THF(100 ml) with heating. The clear solution was brought to roomtemperature and N,N′-disuccinimidyl carbonate (1.92 g, 7.5 mmol) wasadded. DMAP (0.81 g, 7.5 mmol) was added with stirring and the reactionfurther stirred for 16 hours. The solvent was removed under reducedpressure and the residue re-crystallized from n-heptane yielding 1.3 g(68%) of disuccinimidylceramidyl carbonate as white powder m.p. 73-76°C.

(ii) Spermine (0.5 g, 2.5 mmol) and the disuccinimidylceramidylcarbonate (0.39 g, 0.5 mmol) were dissolved in dry dichloromethane withstirring and then treated with catalytic amount of 4-dimethylaminopyridine (DMAP). The solution was stirred at room temperature for 16hours, the solvent evaporated and the residue treated with water,filtered and dried in vacuo, giving 0.4 g (82%) of crude material whichwas further purified by column chromatography on Silica gel, using60:20:20 Butanol:AcOH:H₂O eluent.

(iii) For obtaining a quaternary amine within the compound, the productof step (ii) may be methylated with DMS or CH₃I.

The structure of CCS was confirmed by ¹H- and ¹³C-NMR spectrometry (datanot shown). Detailed description of the analysis is described inco-pending International patent application No. ______.

Other Synthetic Procedures

Similarly to the above procedure, the following procedures may beapplied:

Synthesis of Linear Monosubstituted Ceramde-Spermine Conjugate asDepicted in FIG. 1A

An equivalent of a ceramide is reacted with 2.5 equivalents ofdisuccinimidyl carbonate in the presence of DMAP to obtain thecorresponding 1,3-di-O-succinimidyl derivative is obtained.

The disuccinimidyl derivative though obtained is reacted with anequivalent of spermine at room temperature using catalytic amount ofDMAP to obtain the 3-monosubstituted ceramide-spermine conjugate of FIG.1B.

Synthesis of Linear Disusbstituted Ceramide-Spermine Conjugate asDepicted in FIG. 1B

An equivalent of 1,3-di-O-succinimidyl sphinogid derivative prepared asdescribed above is reacted with 2.5 equivalents of spermine at 800 inthe presence of catalytic amounts of DMAP. The 1,3-disubstituted CCS isthough obtained.

Synthesis of Linear Disusbstituted Ceramide-Branched Spermine Conjugateas Depicted in FIG. 1C

An equivalent of 1,3-di-O-succinimidyl ceramide derivative prepared asdescribed above is reacted with 2.5 equivalents of alpha-omega diprotected spermine at 80° in the presence of catalytic amounts of DMAP.

The protection is removed and the 1,3-“branched” disubstitutedceramide-spermine conjugate is obtained.

Synthesis of Linear Disusbstituted Ceramide-Cyclic Spermine Conjugate asDepicted in FIG. 1D

An equivalent of 1,3-di-O-succinimidyl ceramide derivative prepared asdescribed above is reacted with 0.75 equivalents of spermine at 80° C.in the presence of catalytic amounts of DMAP.

Influenza Antigens

A monovalent subunit antigen preparation derived from influenza A/NewCaledonia/20/99-like (H1N1) strain was generously provided by Drs. Gluckand Zurbriggen, Berna Biotech, Bern, Switzerland. This preparation(designated herein H) comprised of 80-90% hemagglutinin, 5-10 wt %neuraminidase and trace amounts of NP and Ml proteins. A commercialtrivalent subunit vaccine (Fluvirin®) for the 2003/2004 seasoncontaining HN derived from A/New Caledonia/20/99 (H1N1),A/Panama/2007/99 (H3N2) and B/Shangdong/7/97 was obtained from EvansVaccines Ltd., Liverpool, UK. This vaccine was concentrated ˜×8(Eppendorf Concentrator 5301, Eppendorf AG, Hamburg, Germany) prior toencapsulation. A whole inactivated virus was used in some experimentsfor in vitro stimulation.

Lipids

The phospholipids (PL) dimyristoyl phosphatidylcholine (DMPC),dimyristoyl phosphatidylglycerol (DMPG), and dioleoylphosphatidylethanolamine (DOPE) are from Lipoid GmbH, Ludwigshafen,Germany. In addition to DMPC (neutral) and DMPC/DMPG (9/1 mole ratio,anionic) liposomes, 6 formulations of cationic liposomes/lipidassemblies were prepared. The monocationic lipids dimethylaminoethanecarbamoyl cholesterol (DC-Chol),1,2-distearoyl-3-trimethylammonium-propane (chloride salt) (DSTAP),dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), anddimyristoyl-3-trimethylammonium-propane (chloride salt) (DMTAP) are fromAvanti Polar Lipids (Alabaster, Ala., USA). The monocationic lipiddimethyldioctadecylammonium bromide (DDAB) and cholesterol (Chol) arefrom Sigma. The novel, proprietary polycationic sphingolipid N-palmitoylD-erythro sphingosyl carbamoyl-spermine (acetate salt) (ceramidecarbamoyl-spermine, CCS) is from Biolab Ltd., Jerusalem, Israel. Whereindicated, the helper lipids (DOPE, Chol) were used at a lipid/helperratio of 1/1 to 4/1 mole ratio.

Mice

Specific pathogen-free (SPF) female BALB/c mice, 6-8 weeks old, andC57BL/6 mice, 18 month-old, were used (5-10 per group). Animals weremaintained under SPF conditions.

Methods

Encapsulation of Influenza Antigens in Liposomes/Lipid Assemblies

HN antigens (see above) were encapsulated in large (mean diameter 0.1-5μm) heterogeneous (unsized) vesicles. The following procedure was usedroutinely for the preparation of all vaccine formulations. Lipids (10-30mg) were dissolved in 1 ml tertiary butanol, then sterilized byfiltration (GF92, Glasforser, Vorfilter no. 421051, Schleicher &Schuell, Dassel, Germany). The sterile lipid solution was frozen at −70°C., then lyophilized for 24 h to complete dryness. The dried lipidscould be stored at 4° C. for >2 years without significant (<5%) lipiddegradation or loss of “encapsulation” capability. Upon need, the lipidpowder was hydrated with the antigen solution (in PBS pH 7.2) at alipid:antigen (protein) w/w ratio of 3/1 to 800/1. The antigen solutionwas added stepwise in increments of 20-50 III and vortexed vigorouslyafter each addition, up to a final volume of 0.5-1 ml. In someexperiments, the dried lipids were hydrated with PBS and the preformed“empty” lipid assemblies were mixed with the antigen solution. Themixture was vortexed for 1-2 min and used as is within 30-60 min.

To determine “encapsulation” efficiency, two procedures were used,depending on the formulation, resulting in ≧80% separation between thefree antigen and the lipid-associated antigen. For all vaccineformulations, except CCS, the following separation technique was used.The lipid assemblies (1-30 mg lipid) containing the HN antigen (50-100μg protein) were suspended in 0.5 ml PBS and carefully loaded over 0.5ml of D₂O (99.9%, Aldrich Chemical Co., Milwaukee, Wis., USA). Thesample was then centrifuged for 1 h at 30° C. at 45,000 rpm. The free,non-encapsulated HN precipitates while the assembled (liposomal), HN andprotein-free assemblies/liposomes remain in the supernatant. The entiresupernatant was collected and the assemblies/liposomes were dissolved byadding 0.2 ml of warm 10% Triton X-100 to both the supernatant and thepellet fractions. Protein concentration in both fractions was determinedby the modified Lowry technique. For the CCS formulation, the CCS-HN wassuspended in 0.5 ml of PBS-D₂O (1 vol PBS×10+9 vol D₂O) then mixed with0.5 ml of PBS. The mixture was then centrifuged for 10 min at 20° C. at10,000 rpm. The CCS+/−antigen precipitates while the free HN remains inthe supernatant. Lipid dissolution and protein determination in bothfractions were carried out as described above. In both separationtechniques, the overall recovery of the HN antigens was >95%.

Immunization

Free (F-HN) and assembled/liposomal (Lip-HN) vaccines, 0.25-4 μgantigen/strain/dose and 0.075-1.2 mg lipid/dose, were administeredeither once intramuscularly (i.m., in 30 μl), once or twice intranasally(i.n., in 5-50 μl per nostril) spaced 3, 7 or 14 days apart, or twiceorally (in 50 μl) spaced 1 week apart. In all cases, mice were lightlyanesthetized with 0.15 ml of 4% chloral hydrate in PBS givenintraperitoneally. For oral vaccination mice were treated orally with0.5 ml of an antacid solution (8 parts Hanks' balanced salt solution+2parts 7.5% sodium bicarbonate) 30 min prior to vaccination. Choleratoxin (CT, Sigma, USA), 1 μg/dose, was used in all experiments as astandard mucosal adjuvant for comparison. In two experiments, CpG-ODN(ODN 1018, generously provided by Dr. E. Raz, University of California,San Diego, Calif., USA), free and liposomal, 10 μg/dose, was used as anadjuvant.

Assessment of Humoral Responses

Sera, lung homogenates and nasal washes were tested, individually orpooled, 4-6 weeks post-vaccination, starting at 1/10 or 1/20 sampledilution. Hemagglutination inhibiting antibodies were determined by thestandard hemagglutination inhibition (HI) assay, starting at 1/10 sampledilution. Mice with HI titer ≧40 (considered a protective titer inhumans) were defined as seroconverted. Antigen-specific IgG1, IgG2a, IgAand IgE levels were measured by ELISA. The highest sample dilutionyielding absorbance of 0.2 OD above the control (antigen+normal mouseserum, OD<0.1) was considered the ELISA antibody titer.

Assessment of Cellular Responses

Splenocytes obtained at 5-6 weeks after vaccination were tested forproliferative response, IFNγ and IL-4 production, and cytotoxicactivity, following in vitro stimulation with the antigen. Cultures werecarried out at 37° C. in enriched RPMI 1640 or DMEM medium supplementedwith 5% (for proliferation, cytokines) or 10% (for cytotoxicity) fetalcalf serum (FCS), with (for cytotoxicity) or without 5×10⁻⁵M2-mercaptoethanol. Cell cultures were performed as follows: (i)Proliferation: 0.5×10⁶ cells per well were incubated in U-shaped 96-wellplates, in triplicate, with or without the antigen (0.5-5 μg per well),in a final volume of 0.2 ml. After 72-96 h, cultures were pulsed with 1μCi ³H-thymidine for 16 h. Results are expressed in Δcpm=(mean countsper minute of cells cultured with antigen)−(mean counts per minute ofcells cultured without antigen). (ii) Cytokines: 2.5×10⁶ to 5×10⁶ cellsper well were incubated in 24-well plates, in duplicate, with or withoutthe antigen (5-10 μg per well), in a final volume of 1 ml. Supernatantswere collected after 48-72 h and tested by ELISA for murine IFNγ andIL-4 using the Opt EIA Set (Pharmingen, USA). (iii) Cytotoxicity:Responding splenocytes (2.5×10⁶) were incubated as in (ii) for 7 daystogether with an equal number of stimulating BALB/c splenocytes that hadbeen infected with the X/127(H1N1) influenza virus (see below). Forinfection, the splenocytes were incubated, with occasional stirring, for3 h at 37° C. in RPMI 1640 medium (without FCS) with 150hemagglutination units/1×10 ⁶ splenocytes of the virus, followed bywashing. Subsequently, the primed effector cells were restimulated for 5days with infected, irradiated (3,000 rad) splenocytes at aneffector/stimulator cell ratio of 1/4 in the presence of 10 IU/ml ofrhIL-2. Cytotoxicity was measured using the standard 4 h ⁵¹Cr releaseassay at an effector/target cell ratio of 100/1. The labeled targetcells used were unmodified P815 and P815 pulsed for 90 min at 37° C.with the HA2 189-199 peptide (IYSTVASSLVL, 20 μg/1×10⁶ cells).

Determination of Protective Immunity

Mice were anesthetized and 25 μl of live virus suspension per nostril,˜10⁷ EID 50 (egg-infectious dose 50%), was administered, using thereassortant virus X-127 (A/Beijing/262/95 (H1N1)×X-31 (A/HongKong/1/68×A/PR/8/34), which is infectious to mice and cross-reactivewith A/New Caledonia. The lungs were removed on day 4, washed thrice incold PBS, and homogenized in PBS (1.5 ml per lungs per mouse, referredto as 1/10 dilution). Homogenates of each group were pooled andcentrifuged at 2000 rpm for 30 min at 4° C. and the supernatantscollected. Serial 10-fold dilutions were performed and 0.2 ml of eachdilution was injected, in duplicate, into the allantoic sac of11-day-old embryonated chicken eggs. After 48 h at 37° C. and 16 h at 4°C., 0.1 ml of allantoic fluid was removed and checked for viral presenceby hemagglutination (30 min at room temperature) with chickenerythrocytes (0.5 wt. %, 0.1 ml). The lung virus titer is determined asthe highest dilution of lung homogenate producing virus in the allantoicfluid (positive hemagglutination).

Biodistribution and Pharmacokinetics of VariousFluorescently-Labeled-Lipid Formulations and Radioactively-Labeled HNAntigen

Mice were vaccinated once with lissamine-rhodamine labeled lipidassembly formulations either empty or associated with trivalent subunitinfluenza vaccine (HN) in a volume of 20 μl. After 1, 5 or 24 hours,mice were sacrificed and various organs were removed. The organs werestored at −20 deg overnight, and the next morning homogenized in lysisbuffer. 0.2 ml the subsequent homogenate was transferred to eppendorftubes, 0.8 mL of isopropanol was added, and spun for 15 minutes torelease fluorescent probe into the supernatant. 50 uL of the supernatantwas loaded onto a 384 black plate and the fluorescence was read (Em:545, Ex: 596).

In a further assay, 450 μg of trivalent HN vaccine (in 5 mL) weredialysed against DDW (to remove salt) and then concentrated ×1000 to 5μL. The protein was then diluted in 0.1M borate buffer (pH 8.5) to astock solution of 450 μg in 15 μL. The protein was then labeled with¹²⁵I using the Bolton Hunter reagent, according to the manufacturer'sinstructions. Mice were provided with the 125]-labeled HN (2 μg) and at1, 5 and 24 hrs, the mice were sacrificed, and various organs (see FIG.3) were removed into vials and read in a γ-counter calibrated for ¹²⁵I.

The invention will now be defined by the appended claims, the contentsof which are to be read as included within the disclosure of thespecification.

1-75. (canceled)
 76. A method for modulating the immune response of asubject, the method comprises administering to said subject asphingoid-polyalkylamine conjugate together with a biologically activemolecule, the biologically active molecule being effective to modulatesaid immune response.
 77. The method of claim 76, wherein saidsphingoid-polyalkylamine conjugate comprises a sphingoid backbonecarrying, via a carbamoyl bond at least one polyalkylamine chain. 78.The method of claim 76, wherein said modulation includes stimulation orenhancement of the immune response.
 79. The method of any one of claims76, wherein said biologically active molecule is associated with saidsphingoid-polyalkylamine conjugate.
 80. The method of claim 76, whereinsaid biologically active molecule has, at a physiological pH, a netnegative dipole moment, a net negative charge or contains at least oneregion having a net negative charge.
 81. The method of claim 76, whereinsaid biologically active molecule is an immunomodulator selected from anucleic acid molecule, an amino acid molecule or a low molecular weightcompound.
 82. The method claim 76, wherein said biologically activemolecule is selected from an antigenic protein, antigenic peptide,antigenic polypeptide, or a carbohydrate.
 83. The method claim 76,wherein said nucleic acid molecule is an oligodeoxynucleotides (ODN).84. The method of claim 76, further comprising administering to saidsubject an immunostimulating agent.
 85. The method of claim 84, whereinsaid immunostimulating agent is administered concomitant with, or withina time interval before after administration of saidsphingoid-polyalkylamine conjugate.
 86. The method of claim 76, whereinsaid sphingoid-polyalkylamine conjugate forms a lipid assembly.
 87. Themethod of claim 86, wherein said lipid assembly comprises vesicles ormicelles or combination of same.
 88. The method of claim 87, whereinsaid biologically active molecule is associated with said lipidassembly.
 89. The method of claim 76, wherein the sphingoid is selectedfrom ceramide, dihydroceramide, phytoceramide, dihydrophytoceramide,ceramine, dihydroceramine, phytoceramine, dihydrophytoceramine.
 90. Themethod of claim 89, wherein said sphingoid is ceramide.
 91. The methodof claim 90, wherein said polyalkylamine is selected from spermine,spermidine, a polyamine analog or a combination of same thereof.
 92. Themethod of claim 76, wherein said sphingoid-polyalkylamine conjugate isN-palmitoyl D-erythro sphingosyl carbamoyl-spermine (CCS).
 93. Themethod of claim 76, wherein said sphingoid-polyalkylamine conjugate hasthe following formula (I):

wherein R₁ represents a hydrogen, a branched or linear alkyl, aryl,alkylamine, or a group —C(O)R₅; R₂ and R₅ represent, independently, abranched or linear C₁₀-C₂₄ alkyl, alkenyl or polyenyl groups; R₃ and R₄are independently a group —C(O)—NR₆R₇, R₆ and R₇ being the same ordifferent for R₃ and R₄ and represent, independently, a hydrogen, or asaturated or unsaturated branched or linear polyalkylamine, wherein oneor more amine units in said polyalkylamine may be a quaternary ammonium;or R₃ is a hydrogen; or R₃ and R₄ form together with the oxygen atoms towhich they are bound a heterocyclic ring comprising—C(O)—NR₉—[R₈—NR₉]_(m)—C(O)—, R₈ represents a saturated or unsaturatedC₁-C₄ alkyl and R₉ represents a hydrogen or a polyalkylamine of theformula —[R₈—NR₉]_(n)—, wherein said R₉ or each alkylamine unit R₈NR₉may be the same or different in said polyalkylamine; and n and m,represent independently an integer from 1 to 10; W represents a groupselected from —CH═CH—, —CH₂—CH(OH)— or —CH₂—CH₂—.
 94. The method ofclaim 93, wherein R₁ represents a —C(O)R₅ group, R₅ being as defined.95. The method of claim 93, wherein said R₂ and R₅ represent,independently, a linear or branched C₁₂-C₁₈ alkyl or alkenyl groups. 96.The method of claim 93, wherein W represents —CH═CH—.
 97. The method ofclaim 93, wherein R₁ represents a —C(O)R₅ group; R₅ represents a C₁₂-C₁₈linear or branched alkyl or alkenyl; W represents —CH═CH—; R₂ representsa C₁₂-C₁₈ linear or branched alkyl or alkenyl; R₃ and R₄ represent,independently, a group C(O)—NR₆R₇, and R₃ may also represent a hydrogen,wherein R₆ and R₇ represent, independently, a hydrogen or apolyalkylamine having the general formula (II):

R₈—NR₉

_(n)H wherein R₈ represent a C₁-C₄ alkyl; R₉ represents a hydrogen or apolyalkylamine branch of formula (II), said R₈ and R₉ may be the same ordifferent for each alkylamine unit, —R₈NR₉—, in the polyalkylamine offormula (II); and n represents an integer from 3 to
 6. 98. The method ofclaim 93, wherein R₃ is a hydrogen atom.
 99. The method of claim 93,wherein both R₃ and R₄ represent the same or a different polyalkylamine.100. The method of claim 93, wherein R₁ represents a —C(O)R₅ group; R₅represents a C₁₂-C₁₈ linear or branched alkyl or alkenyl; W represents—CH═CH—; R₂ represents a C₁₂-C₁₈ linear or branched alkyl or alkenyl; R₃and R₄ represent independently a group C(O)—NR₆R₇, wherein R₆ and R₇represent, independently, an alkylamine or a polyalkylamine having thegeneral formula (II):

R₈—NR₉

_(n)H wherein R₈ represent a C₁-C₄ alkyl; R₉ represents a hydrogen or apolyalkylamine branch of formula (II), said R₈ and R₉ may be the same ordifferent for each alkylamine unit, —R₈NR₉—, in the polyalkylamine offormula (II); and n represents an integer from 3 to
 6. 101. The methodof claim 93, wherein R₁ represents a C(O)R₅ group; R₅ represents aC₁₂-C₁₈ linear or branched alkyl or alkenyl; W represents —CH═CH—; R₂represents a C₁₂-C₁₈ linear or branched alkyl or alkenyl; R₃ and R₄ formtogether with the oxygen atoms to which they are bonded a heterocyclicring comprising —C(O)—[NH—R₈]_(n)—NH—C(O)—, wherein R₈ represents aC₁-C₄ alkyl, wherein for each alkylamine unit having the formula—NH—R₈—, said R₈ may be the same or different; and n represents aninteger from 3 to
 6. 102. The method of claim 93, wherein said R₈ is aC₃-C₄ alkyl.
 103. The method of claim 76, wherein said biologicallyactive material is derived from influenza virus or an analog of amolecule derived from influenza virus.
 104. The method of claim 103,wherein said biologically active material is a combination ofhemagglutinin and neuraminidase (HN).
 105. The method of claim 76,comprising intranasal or intramuscular administration of said conjugate.106. The method of claim 92, comprising intranasal or intramuscularadministration of said N-palmitoyl D-erythro sphingosylcarbamoyl-spermine together with said biologically active molecule. 107.A method for stimulating or enhancing the immune response of a subjectto influenza virus, the method comprises providing said subject withN-palmitoyl D-erythro sphingosyl carbamoyl-spermine (CCS) together withan influenza antigen.
 108. A vaccine comprising sphingoid-polyalkylamineconjugate and an amount of a biologically active molecule, the amount ofsaid biologically active molecule being effective to modulate the immuneresponse of a subject.
 109. The vaccine of claim 108, wherein saidbiologically active molecule is effective to stimulate or enhance theimmune response of said subject.
 110. The vaccine of claim 109, furthercomprising an immunostimulating agent.
 111. The vaccine of claim 108,wherein said sphingoid-polyalkylamine conjugate comprises a sphingoidbackbone carrying, via a carbamoyl bond at lest one polyalkylaminechain.
 112. The vaccine of claim 111, wherein said sphingoid backbone isselected from ceramide, dihydroceramide, phytoceramide,dihydrophytoceramide, ceramine, dihydroceramine, phytoceramine,dihydrophytoceramine.
 113. The vaccine of claim 112, wherein saidsphingoid is ceramide.
 114. The vaccine of claim 108, wherein saidpolyalkylamine chain is selected from spermine, spermidine or apolyalkylamine analog of spermine or spermidine.
 115. The vaccine ofclaim 108, wherein said sphingoid-polyalkylamine conjugate isN-palmitoyl D-erythro sphingosyl carbamoyl-spermine (CCS).
 116. Thevaccine of claim 115, wherein said biologically active molecule is amolecule derived from influenza virus or is an analog of a moleculederived from influenza virus.
 117. (canceled)
 118. The vaccine of claim33, wherein said sphingoid-polyalkylamine conjugate has the followingformula (I):

wherein R₁ represents a hydrogen, a branched or linear alkyl, aryl,alkylamine, or a group —C(O)R₅; R₂ and R₅ represent, independently, abranched or linear C₁₀-C₂₄ alkyl, alkenyl or polyenyl groups; R₃ and R₄are independently a group —C(O)—NR₆R₇, R₆ and R₇ being the same ordifferent for R₃ and R₄ and represent, independently, a hydrogen, or asaturated or unsaturated branched or linear polyalkylamine, wherein oneor more amine units in said polyalkylamine may be a quaternary ammonium;or R₃ is a hydrogen; or R₃ and R₄ form together with the oxygen atoms towhich they are bound a heterocyclic ring comprising—C(O)—NR₉—[R₈—NR₉]_(m)—C(O)—, R₈ represents a saturated or unsaturatedC₁-C₄ alkyl and R₉ represents a hydrogen or a polyalkylamine of theformula —[R₈—NR₉]_(n)—, wherein said R₉ or each alkylamine unit R₈NR₉may be the same or different in said polyalkylamine; and n and m,represent independently an integer from 1 to 10; W represents a groupselected from —CH═CH—, —CH₂—CH(OH)— or —CH₂—CH₂—.
 119. The vaccine ofclaim 118, wherein R₁ represents a —C(O)R₅ group, R₅ being as defined.120. The vaccine of claim 118, wherein said R₂ and R₅ represent,independently, a linear or branched C₁₂-C₁₈ alkyl or alkenyl groups.121. The vaccine of claim 118, wherein W represents —CH═CH—.
 122. Thevaccine of claim 118, wherein R₁ represents a —C(O)R₅ group; R₅represents a C₁₂-C₁₈ linear or branched alkyl or alkenyl; W represents—CH═CH—; R₂ represents a C₁₂-C₁₈ linear or branched alkyl or alkenyl; R₃and R₄ represent, independently, a group C(O)—NR₆R₇, and R₃ may alsorepresent a hydrogen, wherein R₆ and R₇ represent, independently, ahydrogen or a polyalkylamine having the general formula (II):

R₈—NR₉

_(n)H wherein R₈ represent a C₁-C₄ alkyl; R₉ represents a hydrogen or apolyalkylamine branch of formula (II), said R₈ and R₉ may be the same ordifferent for each alkylamine unit, —R₈NR₉—, in the polyalkylamine offormula (II); and n represents an integer from 3 to
 6. 123. The vaccineof claim 118, wherein R₃ is a hydrogen atom.
 124. The vaccine of claim118, wherein both R₃ and R₄ represent the same or a differentpolyalkylamine.
 125. The vaccine of claim 118, wherein R₁ represents a—C(O)R₅ group; R₅ represents a C₁₂-C₁₈ linear or branched alkyl oralkenyl; W represents —CH═CH—; R₂ represents a C₁₂-C₁₈ linear orbranched alkyl or alkenyl; R₃ and R₄ represent independently a groupC(O)—NR₆R₇, wherein R₆ and R₇ represent, independently, an alkylamine ora polyalkylamine having the general formula (II):

R₈—NR₉

_(n)H wherein R₈ represent a C₁-C₄ alkyl; R₉ represents a hydrogen or apolyalkylamine branch of formula (II), said R₈ and R₉ may be the same ordifferent for each alkylamine unit, —R₈NR₉—, in the polyalkylamine offormula (II); and n represents an integer from 3 to
 6. 126. The vaccineof claim 118, wherein R₁ represents a C(O)R₅ group; R₅ represents aC₁₂-C₁₈ linear or branched alkyl or alkenyl; W represents —CH═CH—; R₂represents a C₁₂-C₁₈ linear or branched alkyl or alkenyl; R₃ and R₄ formtogether with the oxygen atoms to which they are bonded a heterocyclicring comprising —C(O)—[NH—R₈]_(n)—NH—C(O)—, wherein R₈ represents aC₁-C₄ alkyl, wherein for each alkylamine unit having the formula—NH—R₈—, said R₈ may be the same or different; and n represents aninteger from 3 to
 6. 127. The vaccine of claim 118, wherein said R₈ is aC₃-C₄ alkyl. 128-134. (canceled)